Main  Lib. 
«rio.  Dept. 


BIOLOGY 

LIBRARY 

G 


THE    AUTHOH   AT   WORK. 


LABORATORY  METHODS 


OF 


HISTOLOGY  AND  BACTERIOLOGY. 


BY 


J.  H.   HOLMAN,  M.   D., 

Professor  of  Histology  and  Bacteriology,  Meharry  Medical  College; 
Analyst  Mercy  Hospital,  Nashville,  Tenn. 


NASHVItLE,  TENN.: 
NATIONAL  BAPTIST  PUBLISHING  BOARD. 

1903. 


THE  LIBRARY  OF 
CONGRESS, 

Two  Copies  Received 

MAY   18  1903 

Copyright    Entry 


CLASS 

>T 

COPY  A. 


XXc,  No. 

5 


BIOLOGY 

V.'-' 

0 


COPYRIGHT,   1903, 

BY 
JOHN  H.  HODMAN. 


THIS    VOLUME 
IS  RESPECTFULLY  DEDICATED 

TO 

THE    ALUMNI   OF 

MEHARRY    MEDICAL  COLLEGE, 

NASHVILLE, 

TENN. 


268470 


PREFACE. 


This  manual  of  Histology  and  Bacteriology  has  been 
more  particularly  prepared  to  facilitate  the  study  of 
these  important  branches  of  medicine  by  the  author's 
classes.  While  the  author  makes  no  pretension  to  orig- 
inal research  in  this  book,  he  has  endeavored  to  include 
the  useful  and  practical  points  of  the  more  exhaustive 
works  on  the  subject.  In  every  case  the  shortest  and 
best  methods  are  used.  He  has  avoided  technical  terms 
where  practicable  and  has  sought  to  express  the  truth 
in  simple  language.  He  has  provided  blank  sheets  for 
drawing  and  extra  notes  before  and  after  each  part 
of  this  volume. 

The  author  wishes  to  express  his  gratitude  to  friends 
for  encouragement,  especially  to  Dr.  G.  W.  Hubbard, 
M.  D.,  Dean  of  Meharry  Medical  College,  and  Prof. 
W.  Patterson,  A.  M.,  Assistant  Professor  of  Chemistry 
in  the  same  institution.  He  wishes  to  thank  his  as- 
sistant, Mr.  A.  Lyman  Paey,  for  the  illustrations, 
most  of  which  being  original,  having  been  sketched 
from  actual  specimens  prepared  by  the  students  in  the 
laboratory  of  Meharry  Medical  College.  He  also 

(5) 


6  PREFACE. 

wishes  to  express  his  gratitude  to  the  National  Baptist 
Publishing  Board  for  the  excellent  and  artistic  manner 
in  reproducing  the  illustrations  and  getting  out  the 
work. 

The  author  embraces  this  opportunity  to  acknowl- 
edge his  obligations  to  the  following  authorities  for 
information,  viz.:  Sobotta's  Human  Histology,  Bohm 
and  DavidofFs  Histology,  Leroy's  Essentials  of  His- 
tology, and  McFarland.  Eyre  and  Sternberg's  Bacteri- 
ologies. J.  H.  HOLMAN. 

Meharry  Medical  College,  Nashville,  Tenn.,  May, 
1903. 


CONTENTS. 


PART  I. 

Page. 
CHAPTER  I. 

THE  MICROSCOPE  AND  ITS  ACCESSORIES n 

CHAPTER  II. 

MANIPULATION  AND  CARE  OF  THE  MICROSCOPE.  . .   14 
CHAPTER  III. 

GENERAL  HISTOLOGY  26 

CHAPTER   IV. 

THE  DEVELOPMENT  OF  ANIMAL  TISSUE  33 

CHAPTER  V. 

EPITHELIAL  TISSUE 35 

CHAPTER  VI. 

CONNECTIVE  TISSUE  38 

CHAPTER  VII. 

BLOOD  TISSUE   49 

CHAPTER  VIII. 

MUSCLE  TISSUE  56 

CHAPTER  IX. 

THE  VASCULAR  SYSTEM 61 

(7) 


8  CONTENTS. 

Pa^e. 

CHAPTER  X. 
THE  LYMPH  VASCULAR  SYSTEM 65 

CHAPTER  XL 
THE  NERVES 72 

CHAPTER  XII. 
THE  SKIN  AND  ITS  APPENDAGES 85 

CHAPTER  XIII. 
THE  DIGESTIVE  TRACT  95 

CHAPTER  XIV. 
THE  URINARY   TRACT 109 

CHAPTER  XV. 
THE  REPRODUCTIVE  ORGAN 114 

CHAPTER  XVI. 
THE  RESPIRATORY  SYSTEM 120 

CHAPTER  XVII. 

THE  SPECIAL  SENSES 123 

PART  IL 

URINALYSIS    133 

PART  IIL 
BACTERIOLOGY    159 


TECHNIC,  REAGENTS  AND  STAINS 195 


PARTI. 

HISTOLOGY 


LABORATORY     METHODS    OF     HIS- 
TOLOGY  AND    BACTERIOLOGY. 


CHAPTER  L 


THE  MICROSCOPE  AND  ITS  ACCESSORIES. 


The  microscope  is  an  instrument  which  magnifies  an 
object  by  means  of  its  optic  apparatus.  There  are  two 
kinds  of  microscopes,  the  simple  and  compound.  The 
compound  is  used  in  Histology  and  Bacteriology.  The 
parts  and  use  of  the  same  will  be  taught  from  time  to 
time  in  the  Laboratory.  The  base  is  the  part  that 
rests  upon  the  table  and  supports  all  the  rest  of  the 
instrument.  The  pillar  is  the  perpendicular  part  ex- 
tending from  the  base  to  the  back  part  of  the  stage. 
The  arm  is  the  part  that  is  attached  to  the  pillar  and 
works  on  a  hinge- joint.  The  mirror  is  attached  to 
the  arm  in  front  and  works  by  means  of  a  universal 
joint;  its  function  is  to  give  light  to  tn,e  observer 
while  examining  the  object.  The  stage  is  the  plat- 
form upon  which  the  object  rests  while  it  is  being  ex- 

(i-i) 


12  LABORATORY  METHODS  OF 

amined.  The  aperture  is  the  circular  opening  in  the 
stage;  its  diameter  can  be  varied  according  to  the 
amount  of  light  required  to  see  the  object  distinctly  by 
the  use  of  the  diaphragm,  which  is  the  circular  disk 
placed  under  and  attached  to  the  platform.  The  body  is 
the  cylindrical  attachment  attached  to  the  arm  in  front 
and  supporting  the  coarse  adjustment,  draw  tube  and 
objectives.  The  eyepiece  and  the  objectives  are  the 
main  parts  of  the  instrument.  Since  the  eyepiece  and 
objectives  are  the  essential  parts  of  the  instru- 
ment, it  is  very  important  that  we  know  enough 
about  them  to  use  them  intelligently.  The  eye- 
piece consists  of  a  field  glass  and  an  eyeglass, 
which  are  placed  in  the  positions  indicated  by 
their  names.  The  coarse  adjustment  is  placed  on 
the  rear  of  the  arm,  at  the  point  where  the  arm  and 
body  meet.  Its  use  is  to  obtain  a  focus.  The  fine  ad- 
justment is  placed  on  the  top  of  the  arm  and  is  used  to 
complete  the  focus  obtained  by  the  use  of  the  coarse 
adjustment.  The  draw  tube  is  placed  in  the  body  of 
the  instrument  and  is  used  to  increase  its  magnifying: 
power. 

Exercise  No.  i. — Study  of  the  Microscope. 

Make  a  study  of  the  microscope  at  your  locker  and 
learn  to  use  all  the  parts.  Take  the  material  given  you 
and  find  a  focus  of  the  same.  Your  focus  is  the  point 


HISTOLOGY.  13 

where  you  can  see  the  object  best.  Since  you  under- 
stand the  principles  of  the  microscope,  we  will  not 
spend  the  time  to  explain  them,  but  will  use  the  instru- 
ment. 


CHAPTER  IL 


MANIPULATION  AND  CARE  OF  THE  MICRO- 
SCOPE. 


1.  In  taking  an  instrument  from  the  case,  grasp  it 
by  the  base  and  pillar. 

2.  Free  it  from  dust,  with  the  paper  supplied. 

3.  Place  the  instrument  on  the  worktable,  with  the 
opening  of  the  base  from  you. 

4.  Place  your  eye  over  the  eyepiece,  and  at  the  same 
time   manipulate  the   reflector   until  you   get  a   good 
light. 

5.  Place  the  object  to  be  examined  on  the  stage  and 
lower  the  objective  to  a  point  just  above  the  slide  and 
cover-glass,  with  your  head  on  a  level  with  the  stage 
while  doing  so. 

6.  Now  regulate  the  light  by    means    of   the    dia- 
phragm. 

7.  Place  your  eye  over  the  eyepiece  and  rack  up- 
wards until  you  reach  the  point  where  you  can  see  the 
object. 

8.  Note  what  you  see  in  your  notebook. 

9.  Make  drawings  of  what  you  see. 

(14) 


HISTOLOGY.  15 

Handle  the  instrument  with  more  than  ordinary 
care;  never  let  anything  touch  the  lenses.  The  in- 
strument must  be  kept  clean.  Be  careful  and  learn 
to  use  the  instrument  with  both  eyes  open.  Do  not 
vise  it  so  long  that  an  aching  sensation  will  be  pro- 
duced in  the  eyes. 

The  accessories  to  the  microscope  are  many,  but  we 
will  not  use  all  of  them ;  since  we  want  a  working 
knowledge  of  the  following  we  will  learn  their  use: 

The  Substage,  which  is  placed  under  the  stage,  is 
to  support  the  Abbe  condenser. 

The  Iris  diaphragm  is  used  to  regulate  the  light 
which  passes  through  the  condenser. 

The  Abbe  is  a  lens  of  a  very  short  focal  distance  and 
gives  a  very  strong  light. 

The  Mechanical  Stage  is  an  instrument  used  to 
manipulate  the  object  while  upon  the  stage. 

The  Camera  Lucida  is  an  instrument  used  to  assist 
the  student  in  making  the  drawings. 

The  Polariscope  is  used  to  examine  objects  by  means 
of  polarized  light. 

The  Micrometer  is  an  instrument  used  to  determine 
the  magnifying  power  of  the  microscope.  There  are 
two  kinds  of  micrometers,  viz. :  an  eyepiece  and  a  stage 
micrometer.  Determine  the  magnifying  power  of  the 
instrument  at  your  desk. 


16  LABORATORY   METHODS   OP 

1.  Take  the  stage  micrometer  and  place  it  upon 
the  stage  of  the  microscope. 

2.  Build  a  platform  as  high  as  the  stage  of  the  in- 
strument, using  books  for  the  purpose. 

3.  Place  upon  your  platform  a  piece  of  white  paper. 

4.  Now  focus  the  lines  on  the  stage  micrometer, 
keeping  both  eyes  open. 

5.  The  lines  on  the  micrometer  will  be  seen  upon 
the  paper. 

6.  Now  make  a  pinhole  in  the  paper  between  any 
two  lines. 

7.  Multiply  the   distance  between  the  pinholes  in 
the  paper  by  the  denominator  of  the  fraction  on  the 
scale  of  the  micrometer.  The  product  will  be  the  mag- 
nifying power  of  the  instrument. 

The  Microtome  is  an  instrument  that  is  used  to  cut 
tissue  into  very  thin  sections. 

The  Paraffine  Bath  is  an  apparatus  that  is  used  to 
infiltrate  the  tissue  with  paraffine. 

The  Cornet  Forceps  is  an  apparatus  used  to  hold  the 
cover  glass. 

The  Centrifuge  is  an  apparatus  used  to  precipitate 
the  solid  from  a  fluid. 

Slides  and  Cover-Glasses  are  accessories  used  to  re- 
tain or  protect  the  tissue  (or  object). 


HISTOLOGY.  17 

Exercise  No.  2 — Use  the  Polariscope. 

1.  Place  the  microscope  upon  the  table  and  take  off 
the  objective. 

2.  Attach  the  analyzer. 

3.  Reattach  the  objective  to  the  lower  end  of  the 
analyzer. 

4.  Place  upon  the  stage  the  specimen. 

5.  Now  place  upon  the  specimen  the  selenite  plate. 

6.  Now  attach  the  polarizer  and  proceed  to  examine 
the  preparation. 

Use  the  Abbe : 

1.  Place  the  object  upon  the  stage  and  get  a  good 
light. 

2.  Screw  the  Abbe  around  in  place  and  regulate  the 
light  by  the  use  of  the  iris  diaphragm. 

3.  Examine,  note  and  draw. 
Exercise  No.  3. — Use  the  Microtome. 

1.  Place  the  tissue  in  the"  clamps  of  the  instrument 
and  fasten  it. 

2.  Set  your  knife  so  that  it  will  be  at  an  angle  with 
the  tissue. 

3.  Have  the  block  of  tissue  of  the  right  size,  and 
gently  but  firmly  draw  the  knife  across  the  tissue. 


18  LABORATORY  METHODS  OF 

4.  Regulate  the  thickness  of  the  tissue  by  the  use  of 
the  milled  head  on  the  under  surface  of  the  microtome. 
(B.  &  L.  is  the  best  for  students'  use.) 

Exercise  No.  4. — Use  the  Centrifuge. 

1.  Attach  the  instrument  to  the  table  and  place  the 
tubes  in  place. 

2.  Revolve  the  tubes  moderately  for  three  minutes. 

3.  Take  the  sediment  out  and  examine  with  the  mi- 
croscope. 

The  Haematokrit  is  used  in  the  same  way,  only  re- 
volved more  rapidly. 

Exercise  No.  5. — Make  a  spread  of  yeast-plant. 

1.  Obtain  some  yeast  and  place  it  in  a  little  water  in 
a  test  tube ;  set  aside  in  a  warm  place  for  a  short  while. 

2.  Take  a  pipette  and  compress  the  bulb  so  as  to  get 
some  of  the  yeast  and  water  in  the  pipette. 

3.  Now  place  a  small  drop  of  this  material  on  the 
slide  and  examine. 

4.  Examine  with  H.  P.     (1-6  in.  Obj.) 

5.  Observe  the  morphology,  grouping,  budding  and 
search  for  a  chain. 

6.  Make  drawings  and  note  all  you  see. 

This  lesson  is  to  illustrate  the  mode  of  reproduction 
of  cells,  morphology  (or  shape),  the  contents  of  cells, 


HISTOLOGY.  19 

the  function,  and  the  spores  (or  seed  of  cells).  W;e 
use  the  plant  cells  because  they  are  very  large  and  are 
easy  to  obtain  and  possess  all  the  properties  of  the  ani- 


CBLLS  OP  BREWERS'  YEAST. 

1,  A  group  of  two  cells;  2,  dark  spot  represents  a  vacu- 
ole;    cell  forming  a  bud. 

mal  cells.  The  size  of  the  yeast  cells  is  1-4000  to  i- 
2500  of  an  inch  in  diarneter.  Their  form  is  ovoidal. 
They  have  the  power  to  produce  fermentation.  In 
studying  the  plant  under  the  microscope  you  will  do 
well  to  note  whether  there  is  any  chlorophyll  in  the 
plant  or  not.  In  this  way  you  will  be  able  to  make  your 


20  LABORATORY  METHODS  OF 

experiments  of  very  great  value  to  you.  This  work  is  to 
introduce  the  student  to  the  study  of  Histology.  The 
word  Technic  means  the  various  manipulations  neces- 
sary to  get  the  tissue  ready  for  the  microscope. 

The  term  Histology  means  the  microscopic  study  of 
tissues.  The  unit  of  tissues  is  a  cell.  It  is  our  work 
to  investigate  the  relation  that  the  cell  and  intercellular 
substance  bear  to  the  tissues  and  organs  of  the  animal 
or  plant.  Since  we  are  studying  the  human  body  we 
will  confine  ourselves  to  the  study  of  the  tissues  and 
organs  of  animals,  i.  e.,  cat  and  dog,  mostly. 

In  the  beginning  we  will  study  the  cells  of  the  yeast- 
plant,  spirogyra  and  protococcus  in  the  plant  kingdom, 
and  the  green  euglena  and  slipper  animalcule  in  the 
animal  kingdom — each  one  for  a  special  purpose:  the 
euglena,  for  its  property  of  containing  chlorophyll  in 
its  body  and  for  its  possessing  the  power  of  motion; 
the  slipper  animalcule,  for  its  mode  of  reproduction 
and  its  mode  of  locomotion. 

We  will  begin  with  the  yeast  plant  and  go  upwards 
and  see  if  there  are  any  changes  in  the  vegetable  king- 
dom that  resemble  those  in  the  animal  kingdom. 

Exercise  No.  6. — Make  a  spread  of  Protococcus. 

I.  From  the  green  growth  on  a  tree  or  fence,  take  a 
small  bit  of  the  growth  and  place  it  in  a  petri-dish, 
moisten  and  put  in  a  warm  place  for  twelve  hours, 


HISTOLOGY. 


21 


PROTOCOCCUS. 

Showing  morphology,  grouping  and  fission. 

2.  When   the   cells   have  begun   to   vegetate   place 
some  of  them  on  the  slide  and  irrigate  with  water. 

3.  Cover  with  cover-glass  and  examine. 

4.  Use  H.  P.  and  observe  grouping,  cell  contents, 
and  reproduction. 

5.  Irrigate  with  acetic  acid  and  search  for  a  nucleus. 
The  lesson  here  is  to  find  some  reagent  that  will  find 

a  nucleus  without  staining  the  preparation.  Acetic 
acid  is  the  one.  This  is  used  in  all  of  our  work  when 
we  look  for  a  nucleus. 


22  LABORATORY  METHODS  Otf 

Exercise  No.  7. — Make  a  spread  of  scrapings  of  some 
organ  and  examine. 

1.  Take  a  knife  and  scrape  the  surface  of  the  organ 
and  place  the  material  on  a  slide;  cover  and  examine. 

2.  Note  all  you  see,  and  make  drawings. 

Exercise  No.  8. — Make  a  normal  Salt  Soiution.  (NaCl. 
7  grams  to  1,000  c.c.  of  uwter.) 

This  fluid  is  to  be  used  in  all  cases  when  you  want 
to  dilute  the  scrapings. 

Exercise  No.  9. — Make  a  spread  of  Spirogyra. 


SPIROGYRA. 
1,  Showing  mode  of  reproduction;   2,  nucleus. 


HISTOLOGY.  23 

1.  Obtain  from  a  pond  or  brook  a  small  quantity  of 
the  growth  and  place  it  in  a  glass  vessel  with  some 
water. 

2.  Examine  with  H.  P. 

3.  Search  for  nucleus,  note  the  shape  of  the  cells, 
note  the  arrangement  o>f  the  cells  in  the  filament,  ob- 
serve the  arrangement  of  the  chlorophyll  in  the  cells, 
irrigate  with  acetic  and  make  drawings. 

Exercise  No.  10. — Irrigation. 

1.  Place  the  cover  glass  on  the  spread. 

2.  Place  a  drop  of  reagent  on  one  side  of  the  prepa- 
ration and  a  piece  of  blotting  paper  on  the  other. 

3.  Look  through  the  instrument. 

4.  Note  what  you  see  and  draw. 

Any  reagent  may  be  used  for  irrigating.     Irriga- 
tion is  only  used  on  fresh  spreads. 

E.rcrcise  No.  n. — The  Slipper  Animalcule. 

1.  Take  some  of  the  scum  from  the  top  of  the  hay 
infusion. 

2.  Examine  with  H.  P. 

3.  Make  out  all  the  structures  of  the  animal,  note 
and  draw. 


24; 


LABORATORY  METHODS  OF 


SLIPPER  ANIMALCULE. 
1,  Nucleus;   2,  cilia. 

Exercise  No.  12.— Dissociate  some  tendon  of  an  ox. 

i.  Place  some  of  the  tendon  in  enough  weak  soda  so- 
lution to  cover  it  and  let  it  remain  for  two  days. 

Exercise  No.    13.— Tease  some  of  the  tendon  which 
has  been  dissociated  in  the  soda  solution. 

1.  Fix  a  sewing  needle  in  a  pen-holder. 

2.  Use  as  a  teasing  instrument. 

3.  Tear  the  tissue  into  small  bits  and  place  them  on 
the  slide. 


HISTOLOGY.  25 

4.  Examine  with  H.  P.  and  L.  P.     When  using  the 
L.  P.  you  may  dispense  with  the  use  of  the  cover-glass. 


CHAPTER  IIL 


GENERAL  HISTOLOGY. 


The  Cell : — A  cell  is  the  unit  of  all  the  living  organ- 
isms. All  cells  originate  from  a  pre-existing  cell.  A 
complete  cell  consists  of  the  following:  a  cell  wall, 
protoplasm,  nucleus,  nucleolus  and  centrosome.  All 
cells  are  not  complete :  some  may  have  a  nucleus,  and 
some  may  not;  some  may  have  a  cell  wall,  and  some 
may  not.  A  complete  cell  is  called  a  typical  cell,  and 
one  that  is  incomplete  is  called  an  atypical  cell.  The 
cell  body  is  a  mass  of  protoplasm,  having  in  it  a  vary- 
ing amount  of  delicate  fibers  called  "spongioplasm." 
Along  the  course  of  these  we  see  several  little  nodes. 
These  nodes  are  called  microsomes.  That  part  of  the 
protoplasm  near  the  nucleus  is  called  the  nucleoplasm, 
and  that  part  near  the  cell  wall  is  called  the  exoplasm. 
The  power  of  motion  is  located  in  the  protoplasm. 
This  is  illustrated  in  the  Amoeba,  a  small  animal  which 

moves  by  means  of  pseudopods,  i.  e.,  a  part  of  the  pro- 
(26) 


HISTOLOGY. 


:    (  '"  AMOEBA. 

1,  Pseudopods. 

toplasm  flows  to  one  side  of  the  body  of  the  animal, 
and  then  the  rest  of  the  protoplasm  flows  in  the  same 
direction,  until  the  whole  animal  changes  its  position. 

Exercise  No.  14. 

1.  From  the  scum  of  the  hay  infusion  pick  out  a 
submerged  leaf. 

2.  Apply  the  cover-glass  to  the  leaf  and  place  on  the 
slide. 


28  LABORATORY  METHODS  otf 

3.  Examine,  note  the  manner  of  motion  and  sketch 
the  animal  in  several  positions. 

THE  CEXL  WAU,. 

The  cell  wall  is  a  delicate  membrane  surrounding 
the  cell-body,  and  is  derived  from  the  cell-body. 

Exercise  No.  15. — Make  a  microscopic  preparation  of 
Protococcus  and  observe  the  cell  wall. 

The  Nucleus: — This  is  the  highly  refractive  body 
found  in  the  cell-body,  and  is  composed  of  the  nuclear 
membrane,  nuclear  network  and  nucleolus,  seen  only 
under  very  H.  P.  This  can  be  seen  in  the  protococ- 
cus  preparation. 

Centrosomes : — These  are  highly  refractive  bodies 
seen  in  the  nucleus,  and  they  preside  over  the  repro- 
duction of  cells,  while  the  nucleus  transmits  the  hered- 
itary traits  of  the  parent  cell  to  the  new  one. — Leroy. 

The  properties  of  a  cell  are  motion,  reproduction, 
metabolism,  growth,  irritability  and  function.  Metab- 
olism is  that  property  of  a  cell  by  which  it  takes  in 
nutriment  and  assimilates  the  same,  and  the  property 
of  excreting  the  waste  matter,  which  is  the  result  of 
metabolism.  Irritability  is  the  property  of  a  cell 
to  respond  to  nervous  stimulation.  The  function  of 
a  cell  is  the  work  the  cell  will  do.  Reproduction  of  a 


HISTOLOGY.  29 

cell  is  the  power  to  reproduce  a  like  cell.  This  is  ac- 
complished in  two  general  modes :  Direct  and  Indirect. 
The  Direct  is  observed  in  the  protococcus  and  yeast 
plants.  The  Indirect  is  studied  in  the  higher  animals. 
For  convenience  the  stages  of  changes  are  noted  sepa- 
rately. They  are  as  follows :  ( I )  Resting  nucleus ; 
(2)  the  Skein;  (3)  the  Rosette;  (4)  the  Aster;  (5) 
the  Diaster;  (6)  the  Daughter  Rosette;  (7)  the' 
Daughter  Skeins;  (8)  the  Daughter  Nuclei.  Each 
one  of  these  will  be  observed  in  the  experiment  with 
the  growing  tip  of  the  onion. 

Exercise  No.  16. — 

1.  Grow  some  onions  in  a  glass  jar  in  some  water. 

2.  Cut  off  some  of  the  tips  of  the  growing  rootlets. 

3.  Fix  them  in  formalin  solution. 

4.  Harden  in  alcohol  for  I  hour. 

5.  Dehydrate  with  absolute  alcohol. 

6.  Place  the  tissue  in  equal  parts  of  ether  and  abso- 
lute alcohol  for  twelve  hours ;  then  place  it  in  thin  eel- 
loidin  for  twelve  hours. 

7.  Now  place  it  in  thick  celloidin  for  twelve  hours. 

8.  Embed,  section,  mount  and  stain. 

9.  Examine  with  H.  P.  and  the  oil  immersion. 

Technic  for  making  the  solution.     The  formalin  is 
made  as  follows ; 


30  LABORATORY  METHODS  OF 

1.  Take  a  40  per  cent  solution  of  formaldehyd  in 
water  or  alcohol.     Make  a  10  per  cent  solution.     The 
celloidin   solution   is   made    by    taking    5    grams    of 
celloidin  and  100  grams,  i.  e.,  c.c.,   of  the  mixture  of 
equal  parts  of  ether  and  absolute  "alcohol.     Mix. 

2.  The  thick  celloidin  is  made  by  taking  the  same 
mixture  and  adding  celloidin  until  the  mixture  is  as 
thick  as  a  sirup. 

By  sectioning  we  mean  cutting  the  tissue  into  very 
thin  sections,  so  that  they  can  be  easily  made  trans- 
parent, by  the  next  steps  which  are  to  follow.  By 
clearing  a  tissue  we  mean  that  we  make  the  tissue  so 
transparent  that  it  is  of  the  same  transparency  as  that 
of  the  glass  slide  on  which  the  tissue  is  mounted.  Sev- 
eral chemicals  are  used  for  this  purpose.  They  all 
depend  on  their  indices  of  refraction  for  their  value. 
They  are  called  clearing  agents. 

A  piece  of  tissue  to  be  carefully  and  properly 
mounted  must  go  through  the  following  steps : 

1.  The  cells  must  be  killed. 

2.  The  cells  must  be  fixed. 

3.  The  tissue  must  be  hardened. 

4.  The  tissue  must  be  dehydrated. 

5.  The  tissue  must  be  dried. 

6.  The  tissue  must  be  infiltrated. 

7.  The  tissue  must  be  embedded,  :     4 


HISTOLOGY.  31 

8.  Sectioned ;  embeding  material  taken  out ;  then  the 
tissue  fixed  on  the  slide,  stained,  mounted  and  labeled. 
After  the  student  has  carefully  done  this  he  will  not 
have  any  trouble. 

Exercise  No.  17. — 

1.  Applr  to  the  slide  a  section  of  the  outer  skin  of 
an  onion -bulb.     (Apply  the  section  and  add  the  stain.) 

2.  Wash  in  water  to  remove  the  excess  of  stain. 

3.  Dry  with  blotting  paper;  add  a  dehydrating  me- 
dium. 

4.  Wipe  off  the  dehydrating  medium,  which  is  usu- 
ally glycerine. 

5.  Add  xylol. 

6.  Apply  balsam1  to  the  section  and  place  cover-glass 
on  the  section  balsam  downward. 

7.  Label  and  keep  in  a  horizontal  position  until  bal- 
sam hardens   (or  study  at  once).     Make  out  all  you 
can  about  cells  in  this  preparation;  /.  e.,  shape,  size, 
contents  and  motility. 

Exercise  No.  18. — 

Make  a  preparation  similar  to  that  above  and  apply 
a  drop  of  iodine  solution  arid  examine.  Now  remove 
the  cover  and  apply  a  drop  of  sulphuric  acid.  The 


32  LABORATORY  METHODS  OF 

stain  will  take  reddish-brown  ;  then  the  acid  will  attack 
the  cellulose  and  convert  it  into  dextrine;  then  the 
whole  will  be  reddish-brown.  At  first  all  the  starch  will 
be  blue,  due  to  the  presence  of  the  iodine  solution. 
Look  for  Brownian  movement;  this  is  an  independent 
movement  which  is  not  due  to  vitality. 

Exercise  No.    19.  —  Make  a  section  of  a  potato   and 
search  for  starch  granules. 

1.  Place  the  potato  in  the  microtome  and  cut  sec- 
tions 20  microns  thick. 

2.  Place  a  section   on  the  slide  and  search  'for  a 

field  in  which  to  make  observations. 


3.  Note  the  morphology  of  the  cells  and  the  con- 
tents of  the  same. 

4.  Place  a  cover  on  the  section  and  irrigate  with 
iodine  solution. 

5.  Make  notes. 

This  is  the  mode  of  making  a  micro-chemical  ex- 
amination. Note  that  the  morphology  in  this  case  is 
not  the  same  as  in  the  onion  cell, 


CHAPTER  IV. 


THE  DEVELOPMENT   OF   ANIMAL   TISSUE. 


1.  The  cell  or  ovum  of  the  female,  which  is  gener- 
ated in  the  ovary,  passes  down  the  fallopian  tube  and 
meets  the  spermatozoon,  the  male  element,  provided 
conception  has  occurred. 

2.  The   ovum  there  multiplies   by   indirect   division 
into  a  mass  that  resembles  a  mulberry ;  hence  the  mul- 
berry stage. 

3.  The  cells  do  not  all  grow  with  the  same  rapidity, 
so  that  there  are  soon  formed  three  distinct  layers  of 

,  the  original  mass,  i.  e.,  the  cpiblast,  the  hypoblast  and 
the  mesoblast.  The  epiblast  is  the  uppermost  layer, 
the  hypoblast  is  the  under  layer,  and  the  mesoblast  is 
the  middle  layer. 

Now  is  the  time  when  tissue  differentiation  begins. 
All  the  tissues  of  the  body  are  formed  from  these  three 
layers;  hence  the  terms:  mesoblastic,  hypoblastic  and 
epiblastic,  so  often  used  in  medical  literature.  A  tis- 
sue is  composed  of  cells  and  intercellular  substance, 

working  in  common  to  accomplish  some  purpose. 
3  (33) 


34  HISTOLOGY. 

In  this  manual  you  will  find  the  tissue  origin  under 
each  tissue,  as  well  as  on  this  page.  From  the  epiblast 
or  ectoderm  are  derived  the  following  tissues :  nerv- 
ous tissue,  the  epithelium,  covering  the  surfaces  of 
the  body,  the  enamel,  the  nails,  the  hair,  the  organs  of 
special  sense,  and  all  glands,  except  those  which  open 
into  the  alimentary  tract,  from  the  oesophagus  down- 
ward ;  from  the  mesoblast  or  mesoderm  are  derived 
blood,  blood  vessels,  all  the  connective  tissues,  mus- 
cles, dentine  and  cementum ;  from  the  hypoblast  or 
hypoderm  are  derived  the  epithelial  lining  of  the  ali- 
mentary canal  and  the  glands  opening  into  it.  Tissues 
may  be  placed  into  four  groups :  epithelial,  muscular, 
ncri'ous  wid  connective. 


CHAPTER  V. 


EPITHELIAL  TISSUES. 


In    this    manual    Epithelial    Tissue    is    considered 
first. 

Exercise  No.  20. — 


EPITHELIAL  CELLS. 

a,  Squamous;  b,  columnar;  1,  goblet  cells;  a,  ciliated 
epithelial;  1,  cilia;  2,  cell  wall;  3,  nucleus  located  in  the 
protoplasm. 

(35) 


36  LABORATORY  METHODS  OF 

Collect  upon  the  tongue  some  saliva  and  place  it 
on  the  slide;  cover  and  examine  with  H.  P.  Do  you 
see  any  blood  vessels?  Are  the  cells  all  of  the  same 
shape?  What  figure  would  be  the  nearest  to  their 
shape?  What  have  you  concluded  as  to  the  manner 
in  which  they  get  their  nourishment?  Do  they  re- 
semble scales?  If  so,  they  are  of  the  squamous  va- 
riety. There  are  four  varieties  and  it  is  our  purpose  to 
study  them  in  detail.  These  cells  are  reproduced  by 
mitosis.  Give  the  function  of  this  variety  of  cells. 

Exercise  No.  21. — 

1.  Place  upon  the  slide  some  scrapings  from  the 
upper  part  of  the  pharynx  of  a  frog. 

2.  Examine  with  H.  P.  Note  the  following:  shape, 
cilia,  contents  and  size  of  nucleus.     The  functions  of 
the  cilia  is  to  give  motion  to  substances  passing  over 
them.     This  variety  is  columnar  in  shape,  at  the  same 
time  ciliated. 

Exercise  No.  22. — 

Examine  the  scraping  from  the  stomach  of  some 
animal  and  note  the  shape  of  the  cells  there.  This  is 
done  in1  the  same  manner  as  the  others.  You  will  find 
that  the  cells  are  of  a  columnar  variety. 


HISTOLOGY.  37 

Exercise  No.  23. — Make  a  preparation  of  liver  cells 

Do  the  same  as  Exercise  No.  21. 

P.  S. — A  scalpel  is  used  in  scraping. 

Under  the  head  of  Epithelial  there  are  several  modi- 
fied cells.  Some  of  them  contain  mucin  and  others  a 
pigment.  Sometimes  their  shape  takes  on  the  nature 
of  all  the  four  varieties.  They  are  then  called  transi- 
tional epithelial  cells.  Again  they  are  modified  accord- 
ing to  function.  They  are  then  called  specialised  epi- 
thelial cells;  i.  e.,  glandular  and  neuro  epithelial. 


CHAPTER  VI, 


CONNECTIVE  TISSUE. 


Connective  Tissue  is  of  mesodermic  origin.  It 
serves  to  hold  the  different  parts  of  the  body  tog-ether, 
and  contains  more  intercellular  substance  than  the 
epithelial  tissue.  It  serves  to  support  the  organs 
of  the  body  as  well.  There  are  ten  varieties  of  this  tis- 
sue: 


WHITE    FIBROUS    AND    YELLOW    ELASTIC    TISSUES. 

1,  White  fibrous;   2,  yellow  elastic, 
(38) 


HISTOLOGY.  39 

i. — WHITE  FIBROUS  TISSUE. 

Study  the  white  fibrous  tissue. 

1.  Tease  a  piece  of  tendon  of  an  animal  and  place 
it  on  the  slide. 

2.  Stain  with  carmine. 

3.  Observe  the  fibers  running  in  a  wavy  line  across 
the  field.     This  tissue  is  found  in  the  following-  places : 
tendon,  omentum,  subcutaneous  tissue  and  a  few  other 
places. 

II. — YELLOW    ELASTIC   TISSUE. 

Take  the  ligament  of  an  ox's  neck  and  prepare,  stain, 
mount  and  study. 

To  differentiate  yellow  elastic  tissue  from  white  fi- 
brous :  take  a  scalpel  and  cut  each  one.  The  white 
fibrous  will  remain  unaltered,  while  the  yellow  will 
curl  up.  Under  the  microscope  the  white  will  appear 
to  be  in  a  straight  or  wavy  line,  while  the  yellow  will 
appear  to  be  in  branched  processes.  If  you  boil  them 
the  white  will  yield  gelatin,  and  the  yellow  yields  elas- 
tin. 

III. AREOLA   TISSUE. 

It  is  composed  of  white,  fibrous,  yellow  elastic  and 
adipose  tissues,  in  varying  amounts. 


LABORATORY  METHODS  OF 


AREOLA  TISSUE. 

1,   Yellow   elastic   tissue;    2,   white   fibrous   tissue, 
dark  spots  represent  wandering  cells. 


The 


IV. — MUCOUS  TISSUE. 

This  tissue  is  composed  of  large  branching  cells 
surrounded  by  a  soft  gelatinous  substance  (mucin). 
In  embryo  it  is  found  in  the  umbilical  cord.  Study 
this  tissue. 

Technic:  Make  a  spread  of  some  fluid  that  exudes 
from  the  cut  end  of  an  umbilical  cord  and  observe  the 
shape,  size  and  contents  of  the  cells. 


HISTOLOGY.  41 

V. — ADIPOSE  TISSUE. 

This  tissue  is  composed  of  large,  poorly  stained  cells, 
and  is  found  in  a  large  number  of  places  in  the  body. 
These  cells  are  bound  together  by  areola  tissue.  The 
fat  cells  are  derived  from  the  fixed  connective  cells. 


ADIPOSE   TISSUE. 
1,  Nucleus;  2,  protoplasm;   3,  fat  drop;   4,  cell  wall. 

The  protoplasm  is  converted  into  fat,  and  the  nucleus 
is  pushed  to  one -side  and  finally  leaves  the  cell-body. 
Fat  surrounds  every  tissue  of  the  body  except  the  brain 
and  spinal  cord.  This  tissue  is  used  as  a  cushion  for 
the  eye. 


42  LABORATORY  METHODS  OF 

VI.  —  RETlFORM   TISSUE. 


This  variety  of  tissue  is  composed  of  a  number  of 
white  fibers  and  a  varying  amount  of  connective  tissue 
cells. 


RETIPORM    TISSUE. 
Showing  only  the  fibrous  tissue. 

Exercise  No.  24. — 

Take  a  section  of  a  fresh  spleen  and  shake  it  in  an 
indifferent  fluid  for  several  minutes;  then  place  it  on 
the  slide  and  examine.  An  indifferent  fluid  is  a  fluid 
that  has  no  effect  on  the  tissue  which  is  placed  in  it, 


HISTOLOGY. 


43 


i  VII. — ADENOID  TISSUE. 

This  tissue  is  simply  retiform  tissue  having  a  vary- 
ing amount  of  lymphoid  cells  entangled  in  its  meshes. 

VIII. — CARTILAGE   TISSUE. 

This  is  a  variety  of  connective  tissue  that  is  widely 
distributed.  There  are  three  main  varieties  of  carti- 
lage ;  viz. : 


WHITE  FIBRO-CARTILAGE. 

1,  White  fibers;   2,  cartilage  cells;   1,  2,  3,  4,  cartilage 
cells  in  different  stages  of  reproduction. 


44  LABORATORY  METHODS  OF 

White  Fibro-Cartilage. — This  variety  is  composed 
of  the  following  material:  matrix,  capsule,  peri- 
chondrium,  cells  and  lacunae.  The  matrix  has  em- 
bedded in  it  a  few  white  fibers :  hence  the  name.  The 
matrix  is  a  homogeneous  material,  semisolid  in  con- 
sistency, and  will  yield  chondrin  on  boiling.  The  oval 
shaped  cells  lie  embedded  all  in  the  matrix,  in  groups 
of  two,  four  and  eight,  etc.  The  cells  are  those  oval 
bodies  scattered  all  in  the  matrix.  The  lacunae  are 
those  hardened  areas  found  near  the  cells  on  all  sides, 
those  white  threads  that  run  all  in  between  the  cells 
and  give  the  fibrous  nature  to  the  cartilage.  The  peri- 
chondrium  is  the  membrane  that  is  found  on  the  out- 
side of  the  cartilage,  and  is  composed  of  two  layers: 
one  to  protect  the  cartilage;  the  other  to  furnish  new 
cells  in  the  growing  cartilage. 

Hyaline  Cartilage  is  composed  of.  a  matrix,  peri- 
chondrium,  cells  and  lacunae. 

Exercise  No.  25. — Mount  a  T.  $.  of  trachea  of  a  cat; 
stain  unth  carmine. 

1.  Cut  the  section  with  the  microtome  20  microns 
thick. 

2.  Place  the  section  on  the  slide  and  stain. 

3.  Wash  off  excess  O'f  stain  with  acid  alcohol. 

4.  Wash  in  70  per  cent,  alcohol;  then  80  per  cent, 
and  90  per  cent, 


HISTOLOGY. 

5.  Wash  in  100  per  cent. 

6.  Clear  up  with  some  of  the  clearing  agents. 

7.  Dry  and  mount  in  balsam. 

T.  S.  is  the  abbreviation  for  transverse  section. 


45 


FIBRO   ELASTIC    CARTILAGE. 

The  Fitiro  Elastic  Cartilage. — This  variety  of  tissue 
is  composed  of  yellow  elastic  tissue  in  addition  to  the 
material  in  the  white  fibrous  cartilage. 

IX  —BONE  TISSUE.  **w' 

This  variety  of  tissue  gives  shape  to  the  body  and  is 
therefore  very  hard.  It  is  composed  of  the  following 


46  LABORATORY  METHODS  OF 


T.  S.  BONE. 
1,  Haversian  canal;  2,  lacunae;  3,  canaliculi. 

parts :  H aversion  system,  Haversian  canals,  latmellae, 
lacunae,  canaliculi  and  bone  cells.  The  Haversian  sys- 
tem is  composed  of  Haversian  canals,  a  number  of 
canaliculi,  a  number  of  lamellae  and  a  number  of  bone 
cells.  The  cells  lie  in  the  bone  matrix  in  much  the 
same  way  as  they  do  in  the  cartilage  matrix.  The 
lacunae  are  also  very  much  the  same.  The  bone  cells 
send  out  processes  for  the  purpose  of  conveying-  nutri- 
ment to  all  parts  of  the  bone ;  these  processes  are  called 
canaliculi.  The  Haversian  canals  cafry  the  small 


HISTOLOGY.  47 

blood  vessels  into  the  bones.  The  lamellae,  arranged 
around  the  Haversian  canal,  are  called  the  Haversian 
lamellae.  The  lamellae  running  parallel  with  the  outer 
surface  of  the  bone  are  called  the  ground  lamellae,  and 
those  running  between  the  Haversian  system,  are  called 
the  interstitial  lamellae.  There  are  little  canals  run- 
ning from  the  periosteum  to  the  nearest  Haversian  la- 
mellae. These  canals  are  called  Volkmann's  canals. 
Occasionally  uncalcified  lines  are  seen  passing  ob- 
liquely or  transversely  through  the  lamellae.  These, 
are  called  the  lines  of  Sharpey.  The  periosteum 
consists  of  fibrous  connective  tissue,  and  is  divided 
into  two  layers.  One  layer  is  the  bone-forming 
layer  and  the  other  is  the  bone-protecting  layer.  Bone- 
marrow  is  composed  of  two  kinds  of  cells,  the  red  and 
yellow.  The  yellow  is  mostly  of  the  fat  variety,  and 
the  red  is  mostly  of  red  blood  cells.  The  blood  vessels 
of  the  bone  tissue  are  distributed  through  the  Haver- 
sian system  and  the  canals  of  Volkmann.  The  perios- 
teum is  richly  supplied  with  blood  vessels. 

Bone  develops  in  two  ways :  centers  of  ossification 
and  perichondrial.  The  perichondrial  is  as  follows: 
the  cells  of  the  immature  bone  pass  down  the  lines  of 
Volkmann  and  locate  themselves  in  a  center  called  a 
center  of  ossification.  Ossification  means  the  depo- 


48  '  HISTOLOGY. 

sition  of  salts  of  lime  in  the  tissues.  These  salts  are 
usually  deposited  in  cartilage,  and  then  it  is  called 
bone.  Dentine  is  a  variety  of  bone,  and  .will  be  dis- 
cussed more  fully  when  we  come  to  the  subject  of  the 
teeth. 


No.  26.  —  Make   a   preparation    of    cartilage 
from  the  end  of  bone  in  the  fresh  state. 

1.  Take  a  sharp  knife  and  cut  a  piece  of  cartilage 
from  the  end  of  a  long  bone  and  section. 

2.  Mount  in  salt  solution. 

3.  Stain. 

4.  Study,  draw  and  note. 

Look  for  the  following:  matrix,  cells,  capsule,  lacu- 
nae and  perichondrium. 

Exercise  No.  27.  —  Examine  the  L.  $.  of  bone  given 
you. 

Look  for  the  following:     Haversian  system,  Haver- 
sian  canal,  lacunae,  bone  cell  and  canaliculi. 

Exercise  No.  28.  —  Examine  the  T.  S.  section  of  the 
bone  given  you. 

Look  for  the  same  as  you  did  in  the  previous  exer- 
cise.    Make  drawings  in  both  cases  ;  note  and  study. 

Exercise  No.  29.  —  Examine  the  cuts  in  your  text  and 
make  a.  draining  of  all  you  see. 


CHAPTER  VIL 


BLOOD  TISSUE. 

[The  tenth  variety  of  Connective  Tissue  mentioned  in 
this  work.] 

Blood  is  a  tissue  of  mesodermic  origin,  and  has  n 
fluid  intercellular  substance,  known  as  plasma.  The 
cellular  elements  are  many,  viz. :  white  corpuscles,  red 
cells,  and  the  platelets.  The  blood  is  distributed  to 
all  parts  of  the  body  by  means  of  tubes  called  arteries, 
and  is  returned  to  the  heart  by  means  of  the  veins. 
The  capillaries  are  the  terminals  of  the  arteries  and  the 
beginning  of  the  veins.  Their  walls  are  very  thin,  and 
admit  a  change  of  gases  between  the  blood  and  the 
tissues. 

Exercise  No,  30. — Obtain  a  drop  of  blood  from  your 
finger. 

1.  Sterilize  the  lance  and  prick  the  part  selected. 

2.  Wipe  off  the  first  drop  of  blood  with  a  clean  cloth 
wet  in  alcohol. 

4  (49) 


50  LABORATORY  METHODS  OF 

3.  Constrict  the  part  above  the  puncture  and  obtain 
another  drop. 

4.  Place  this  drop  on  the  slide  and  cover,  or  touch 
the  edge  of  two  clean  cover-glasses  to  the  drop,  so  that 
a  very  thin  spread  of  blood  can  be  secured  between 
them;  place  on  the  slide  and  examine. 

5.  Note  the  shape  of  the  red  cells;  the  manner  of 
grouping  on  the  slide ;  search  for  nuclei ;  look  for  stain 
in  the  cells ;  note  the  central  part  of  the  cell,  and  meas- 
ure the  cell  with  the  micrometer. 

6.  In  the  same  preparation  examine  the  white  cor- 
puscles which  are  found  adhering  to  the  slide. 

Exercise  No.  31. — Use  the  Haemacytometer,  count  the 
cells  of  both  varieties,  in  a  c.m. 

1.  Take  995  c.m.  of  sodium  sulphate  solution;  spe- 
cific gravity,  1028;  temperature,  15  C. 

2.  Take  5  c.m.  of  the  blood. 

3.  Mix  in  the  mixing  jar  with  the  spud. 

4.  Take  the  instrument  and  fill  the  cell  with  the 
mixed  blood  and  solution. 

5.  Place  the  instrument  under  the  microscope  and 
let  the  whole  stand  a  few  minutes ;  then  count  the  num- 
ber of  cells  in  ten  squares;  strike  the  average;  then 
multiply  the  average  by  10,000,  since  the  blood  has 


HISTOLOGJ.  51 

been  diluted  and  since  the  squares  are  only  one-tenth 
of  a  m.m.  square.  The  average  number  of  red  cells 
is  about  5,000,000  in  each  c.m.  of  blood  in  men,  and 
4,500,000  in  women. 

Exercise  No.  32. — Use  the  Haemoglobinometer. 

1.  Take  the  instrument  and  set  it  up  in  front  of  a 
piece  of  white  paper. 

2.  Obtain  20  c.m.  of  blood  and  place  it  in  the  tube 
provided  for  the  reception  of  the  blood. 

3.  Dilute  the  blood  with  water  until  the  blood  mix- 
ture and  the  standard  mixture  in  the  tube  coincide  in 
color. 

If  the  blood  has  the  required  amount  of  haemoglobin 
in  it,  when  you  have  added  the  water  up  to  the  gradu- 
ate 100,  the  two  colors  will  coincide.  If  it  reaches  the 
color  before  the  required  amount  of  w&ter  has  been 
added,  there  is  not  enough  haemoglobin  in  the  speci- 
men;  and  if  it  requires  more  water  than  the  100,  there 
is  too  much  haemoglobin. 

Exercise  No.  33. — Use  the  Haematokrit. 

I.  Set  up  the  centrifuge  and  place  the  Haematokrit 
in  place;  revolve  the  centrifuge  very  rapidly  for  three 
minutes. 


52  LABORATORY  METHODS  OF 

2.  Observe  the  position  of  the  heavier  materials  in 
the  blood. 

3.  Hold  the  instrument  between  you  and  the  light; 
see  how   far  the  red  cells   register  on  the  graduate. 
This  is  a  tube  of  small  bore  and  is  arbitrarily  gradu- 
ated.    It  registers  per  cent. 

Exercise  No.  34.— Make  a  spread  of  frog's  blood. 

i.  Snip  the  nose  of  the  animal  with  a  sharp  instru- 
ment and  obtain  a  small  drop  of  blood. 


FROG'S  BLOOD. 
1,  Leukocytes;  2,  oval  red  cells. 


HISTOLOGY.  53 

2.  Collect  it  in  the  same  way  that  you  did  the  blood 
of  man. 

3.  Examine  the  shape  of  the  corpuscles ;  search  for 
a  nucleus  in  the  red  corpuscles;  measure  the  cells  in 
both  diameters,  and  examine  the  white  corpuscles  for 
the  amoeboid  movement. 

Exercise  No.  35. — Make  a  spread  of  human  blood  and 

apply    the   reagents  given  you,   viz.:  salt  solution, 

acetic  acid,  and  water. 

Irrigate  in  the  usual  way.  In  the  salt  preparation 
r  - 1  e  what  you  see  ;  give  the  cause.  Do  the  same  with 
all  the  others.  Do  red  cells  have  a  cell-wall?  (Leroy 
and  Sobotta  say  they  do>. 

The  colorless  corpuscles  are  of  five  varieties:  [mono- 
nuclear  lymphocytes,  poly  nuclear  lymphocytes,  large 
lymphocytes,  eosinophiles  and  basophiles.  These  are 
all  larger  than  the  red  blood  cells.  They  all  originate 
from  the  lymph  glands.  They  are  separated  into  their 
classes  by  the  use  of  different  stains,  and  the  use  of  the 
eye-piece  micrometer.  A  large  per  cent  of  them  has 
the  power  oi  moving  like  the  ameba;  hence  the  term 
ameboid  movement,  as  applied  to  the  white  blood  cells. 
Blood  shadows  are  red  blood  cells  that  have  been  sub- 
jected to  the  action  of  water,  and  have  the  haemoglo- 
bin dissolved  out,  leaving  the  shadow  of  the  cell.  Blood 
platelets  are  small  red  cells.  The  crystals  found  in 


54 


LABORATORY  METHODS 


blood  are  haemoglobin,  haematin,  haematoidin.  The 
most  important  are  the  hemin,  which  can  be  demon- 
strated by  the  following  method : 


HUMAN  BLOOD. 

1,  Biconcave  red  blood  cells;  2,  red  blood  cells  on  edge; 
3,  spherical  red  blood  cells;  4,  red  Hood  cells  after  the  ac- 
tion of  water;  5,  leukocytes;  6,  rouleaux.  Crenated  red 
cells  to  left  of  the  rouleaux. 

Obtain  a  drop  of  blood  in  the  usual  way,  and  apply 
to  it  some  salt  solution  and  some  acetic  aicid;  let  the 
whole  dry  for  a  few  minutes.  Examine  with  H.  P. 

The  red  blood  cells  are  derived  from  the  mesoblast, 
and  are  nucleated  and  colorless  when  first  formed. 


HISTOLOGY.  55 

These  multiply  and  lose  their  nuclei  and  acquire  hemo- 
globin. Later  in  life  the  spleen  and  red  marrow  of  the 
bone  contribute  to  the  supply  of  red  cells.  The  white 
blood  cells  are  derived  mostly  from  the  lymphoid  tis- 
sue and  spleen.  The  lymphoid  corpuscles  become  dis- 
lodged and  carried  along  the  blood  current  or  lymph 
current,  as  the  case  may  be.  "In  these  places  they  un- 
dergo a  mature  change." — Lcroy. 

Exercise    No.    36. — Make    a    stained    preparation    of 
blood. 

1.  Make  a  spread  in  the  usual  way. 

2.  Fix  high  over  the  flame. 

3.  Apply  eosin  30  minutes. 

4.  Haematoxylin   3   minutes. 

5.  Dry,  mount  and  label. 

6.  Study  and  preserve, 

Search   for  the  four  varieties  of  lymphocytes  and 
observe  the  formation  of  rouleaux  in  the  red  cells. 


CHAPTER  VIII 


MUSCLE  TISSUE. 


This  variety  of  tissue  is  of  mesoblastic  origin,  and  is 
divided  into  three  classes :  voluntary,  involuntary  and 
heart  muscle.  All  muscle  tissue  is  rich  in  blood  vessels, 
lymphatics  and  nerves.  The  blood  vessels  break  up 
into  capillaries,  which  form  a  net  work  around  each 
individual  fiber.  The  nerves  terminate  in  special  end- 
organs  under  the  sarcolemma. 

The  voluntary  or  skeletal  muscle  is  the  most  wide- 
ly distributed.  It  forms  all  the  skeletal  muscles,  and 
is  under  the  control  of  the  will.  It  is  composed  of 
long,  cylindrical  fibers,  by  some  authors  called  cells. 
These  are  the  largest  cells  in  the  body — so  large  that 
one  cannot  be  placed  in  the  field  of  the  microscope  at 
once.  Sometimes  they  are  il/2  inches  long.  They 
are  all  surrounded  by  a  delicate  membrane,  or  cell-wall, 
called  sarcolemma,  just  under  which  are  placed  the 
nuclei  of  the  cell.  The  transverse  striations  that  are 
seen  all  along  the  course,  are  said  to  be  due  to  the  pres- 
ence rnd  make-up  of  the  sarcous  elements.  There  are 
seen  running  along  the  long  diameter  of  the  cell  deli- 
(56) 


HISTOLOGY. 


57 


cate  lines,  which  are  called  sarcostyles  and  can  be 
demonstrated  by  taking  a  piece  of  the  tissue  and  disso- 
ciating the  same.  This  will  separate  the  lines  into  in- 
dividual fibrils.  Each  fibril  is  called  a  sarcostyle.  The 
space  between  the  sarcous  elements  is  filled  with  sub- 
stance, which  is  called  sarcoplasm,  and  is  analogous 
to  the  protoplasm  in  other  cells.  This  protoplasm  is 
endowed  with  the  special  power  of  motion,  or  con- 
tractility. If  we  use  a  very  H.  P.  we  will  be  able 


SECTION    OF   VOLUNTARY   MUSCLE. 

A.  Fiber  or  Cell.     1.  Sarcolemma;    2,  fibrillae. 

B.  Two  fibers,  one  showing  torn  sarcolemma  as  a  clear 

space. 

C.  A  sarcostyle;    1,  Fusiform  body;    2,  a  ball;    3,  lighc 

spaces. 


58  LABORATORY  METHODS  OP 

to  observe  the  lines  of  Hensen  and  the  membrane  of 
Krause.  These  lines  are  located  in  the  sarcous  ele- 
ments. The  sarcous  elements  are  composed  of  the  fol- 
lowing parts,  viz. :  a  spindle-shaped  body,  light  space, 
and  a  bill.  Placed  end  to  end  they  make  a  fiber  or  cell. 
The  lines  of  Hensen  are  those  lines  seen  dividing  the 
spindle-shaped  bodies,  and  the  membrane  of  Krause  is 
the  membrane  or  line  drawn  across  the  light  bands. 
The  following  wrappings  are  found  enveloping  the 
muscle:  endomysium,  the  membrane  around  the  fiber. 
A  number  of  fibers  (wrapped  in  the  endomysium)  is 
put  together  and  forms  a  bundle;  this  bundle  is 
wrapped  in  another  membrane,  called  a  perimysium.  A 
number  of  these  bundles  is  put  together  and  enveloped 
in  another  membrane,  called  epimysium.' 

Involuntary  Muscle. — This  variety  of  tissue  is  net 
under  the  control  of  the  will.  The  cells  are  fusiform 
in  shape,  and  have  their  nuclei  centrally  located.  They 
are  joined  together  by  overlapping  each  other;  ait  the 
point  where  the  cells  meet  a  cement  substance  is  seen. 
They  are  found  in  the  tissues  that  are  not  under  the 
control  of  the  will,  i.  e.,  the  intestines,  uterus,  and  rec- 
tum. 

Heart  Muscle. — This  variety  of  muscle  is  found  only 
in  the  heart,  and  is  not  the  same  as  the  involuntary 
muscle,  in  shape  or  function.  The  cells  are  short,  rec- 


HISTOLOGY.  59 

tangular  shape,  with  branching  interlacings,  as  well 
as  a  cement  substance  between  the  cells.  With  a  H.  P. 
can  be  seen  a  lairge  number  of  fine  transverse  striations 


L.  S.  HEART  MUSCLE. 

in  each  cell;  also  a  number  of  longitudinal  lines  are 
seen  in  them.  The  cell  has  a  single  nucleus  centrally 
located.  Between  each  one  is  found  the  capillaries 
and  fibrous  tissue,  which  serves  to  hold  the  tissue  in 
place. 

Exercise  No.  37. — Mount  some  fresh  muscle  tissue. 

Take  some  teased  muscle,  place  it  on  the  slide  and 
examine.     Search  for  the  following  parts,  and  make 


£0  HlSTOLOGt. 

drawing:  cells,  nuclei,  and  the  enveloping  membranes. 
Proceed  in  like  manner  with  all  three  varieties. 

Exercise  No.  38. — Mount  the  prepared  tissue  supplied, 
and  study,  cover,  label  and  preserve. 


CHAPTER  IX. 


THE  VASCULAR  SYSTEM. 


The  vascular  system  is  composed  of  the  heart,  ar- 
teries, veins  and  capillaries.  The  heart  is  a  muscular 
organ,  lined  by  an  endocardium,  which  is  the  continua- 
tion of  the  tunica  intima  of  the  blood  vessels.  Next 
to  the  endocardium  is  the  myocardium  from  within 
out.  The  myocardium  is  the  heart  muscle.  The  outer 
surface  of  the  heart  is  covered  by  the  pericardium: 
this  is  a  serous  membrane,  of  white  fibrous  tissue,  and 
covered  by  endothelial  cells.  Numerous  ganglia  are 
found  in  the  tissues  of  the  heart,  along  the  course  of 
the  nerves.  The  nerve  supply  of  the  heart  is  derived 
from  the  pneumogastric  and  sympathetic  system.  The 
fibers  running  from  these  centers  are  both  medullated 
and  non-medullated.  The  first  is  from  the  pneumo- 
gastric, and  the  second  is  from  the  sympathetic  sys- 
tem. The  valves  of  the  heart  are  simply  broad  folds 
of  the  endocardium,  between*  which  are  a  small  amount 

(61) 


62  LABORATORY  METHODS  OP 

of  fibrous  tissue.  The  lymphatics  of  the  heart  are 
many  and  are  found  in  two  places,  viz. :  around  the 
coronary  arteries  and  between  the  muscle  fibers  The 
capillaries  are  minute  ramified  arteries,  which  have  lost 
their  outer  coat  and  hence  become  very  thin.  This 
thin  space  is  where  the  white  blood  cells  leave  the  ves- 
sels in  inflammation;  the  spaces  are  called  stigmata. 
The  blood  vessels  are  richly  supplied  with  lymphatics 
which  are  seen  ramifying  in  the  outer  coat  of  the 
larger  arteries  and  surrounding  the  smaller  capillaries. 


T.  S.  BLOOD  VESSEL. 
Showing  bands  of  yellow  elastic  tissue. 
Tunica  intima;  2,  Tunica  media;   3,  Tunica 


HISTOLOGY.  63 

The  veins  are  different  from  the  arteries  in  that  they 
have  thinner  coats  and  less  muscle  tissue;  their  walls 
are  thinner  than  the  arteries  and  their  lumen  is  larger. 
They  have  more  fibrous  tissue  than  the  arteries.  Ar- 
teries are  composed  of  three  coats.  The  tunica  intiw*, 
the  tunica  media,  and  the  tunica  adventitia.  The  in- 
ternal coat  is  lined  with  endothelial  cells  resting  upon 
a  basement  membrane  of  white  fibrous  tissue.  Just 
under  this  coat  is  located  a  band  of  yellow  elastic 
tissue,  called  the  fenestrated  membrane  of  Henle. 
The  middle  coat  is  composed  of  the  smooth  muscle 
running  around  the  artery,  and  having  a  few  fibers  of 
fibrous  tissue  interspersed  with  the  muscle  cells,  es- 
pecially the  large  arteries.  The  outer  coat  is  com- 
posed of  white  and  yellow  elastic  tissue  in  abundance. 
The  nerve  supply  of  the  artery  is  derived  from  the 
vasomotor  system,  and  the  blood  supply  of  a  part  is 
regulated  by  the  contraction  or  expansion  of  the  mus- 
cle fibers  in  the  middle  coat  of  the  artery.  The  outer 
coat  is  for  strength ;  the  inner  one  for  smoothness ;  the 
middle  one  for  contraction  and  expansion.  All  the 
blood  vessels  are  of  mesoblastic  origin.  The  vessels 
are  closed  tubes. 

Exercise  No.  39. — Make  a  section  of  a  blood  vessel^ 
mount,  stain  and  preserve. 


54  HISTOLOGY. 

Technic:  Use  the  alcohol  hardening  method.     Ob- 
serve the  coats,  and  make  out  the  yellow  elastic  tissue. 

Exercise  No.  40. — Make  a  stained  preparation  of  an 
artery  and  vein. 


CHAPTER  X. 

THE  LYMPH  VASCULAR  SYSTEM. 

The  lymphatics  are  in  all  particulars  like  the  blood 
vessels  in  structure,  .except  that  even  the  largest 
lympatics  have  only  a  small  amount  of  muscle  tissue. 
In  all  the  tissues  and  organs  there  are  small  spaces  that 
are  called  pericellular  spaces.  These  spaces  are  the 
beginning  of  lymphatic  vessels.  The  small  vessels 
continue  to  join  until  they  reach  the  thoraic  duct,  which 
pours  its  contents  into  the  sub-clavian  vein :  at  this 
point  the  inner  court  of  the  duct  is  folded  over  on  it- 
self and  forms  a  valve  to  prevent  blood  from  flowing 
into  the  duct.  All  along  the  course  of  the  vessels  are 
found  similar  valves,  which  prevent  the  lymph  from 
flowing  but  one  way.  While  all  the  tissues  of  the  body- 
are  rich  in  lymph  vessels,  the  ones  supplied  most  are 
the  white  fibrous,  areola,  blood  vessels  and  nerves.  In 
the  last  two  locations  they  are  called  perivascular  and 
perineurial  lymphatics,  respectively.  The  lymph  is  a 
clear  fluid,  having  floating  in  it  a  large  number  of 
cells  and  fat  granules.  The  cells  are  very  much  like 
5  (65) 


66  LABORATORY   METHODS   OF 

the  small  lymphocytes  of  the  blood.  The  fat  is  al- 
ways present  in  varying  amounts,  but  is  increased  after 
a  meal.  The  cells  of  the  lymph  eventually  become 
the  white  blood  cells. — Osbnrn. 

DIFFUSE   ADENOID   TISSUE. 

In  many  positions  throughout  the  body  there  are 
found  small  nodes  oi  adenoid  tissue  located  in  the 
tunica  propria  of  mucous  membranes,  as  in  the  lungs 
and  digestive  tract.  This  variety  of  adenoid  tissue  is 
called  diffused.  In  some  places  this  tissue  is  present 
in  small  amounts,  and  fades  away  into  the  surrounding 
tissue  without  presenting  any  line  of  demarcation.  In 
other  locations  this  tissue  is  surrounded  by  a  band  of 
white  fibrous  tissue,  having  dispersed  it  in  a  few 
smooth  muscle  fibers.  This  band  is  called  a  capsule. 
The  node  and  band  is  called  a  lymph-follicle.  When 
these  nodes  occur  in  the  course  of  a  lymph  vessel,  they 
are  called  lymph  glands.  These  glands  are  usually 
bean-shaped  and  the  capsule  sends  down  little  bands  of 
white  fibrous  tissue,  separating  the  gland  into  divisions. 
These  bands  are  called  trabecula,  and  the  divisions, 
lobules.  The  trabeculae  subdivide,  forming  the  reti- 
form  tissue  of  the  gland,  in  which  are  found  the  Mal- 
pighi?n  corpuscles  of  the  lymph  glands.  The  blood 
vessels  enter  the  capsules.  The  nerves  of  these  glands 


HISTOLOGY. 


67 


are  of  both  varieties,  viz. :  medullated  and  non-medul- 
lated.  That  portion  of  the  gland  which  is  located  near 
the  capsule  is  called  the  cortical  portion,  and  that  por- 
tion near  the  center  of  the  gland  is  called  the  medul- 
lary portion. 

THE  SPLEEN. 

The  spleen  is  a  lymphoid  gland,  and  differs  from 
the  other  lymphoid  glands  in  that  it  has  no  medullary 
portion  and  the  blood  supply  is  differently  arranged. 


SECTION  OF  SPLEEN. 

1, -Capsule;  2,  Malpighian  corpuscles;  3,  intralobular 
vein;  4,  splenic  pulp.  The  large  bands  of  fibrous  tissue 
represent  the  trabeculae. 


68  LABORATORY  METHODS  OF 

The  splenic  artery  enters  the  spleen  at  the  hilum 
and  passes  up  the  trabeculae  for  a  certain  distance, 
and  then  leaves  them  and  passes  into  the  splenic 
pulp ;  here  the  artery  loses  its  outer  coat  and  con- 
tinues as  the  lymphoid  tissue  of  the  spleen.  In  this 
place  the  adenoid  tissue  collects  into  a  dense  mass  and 
the  mass  is  called  the  Malpighian  corpuscle  of  the 
spleen.  Here  the  germ  center  is  found.  The  veins 
begin  where  the  artery  ends  and  pass  to  the  trabeculae, 
where  they  pass  out  of  the  organ  by  following  the  tra- 
beculae to  the  hilum.  A  few  lymphatics  are  found  in 
the  capsule  and  trabeculae.  The  nerves  of  the  glands 
are  few  in  number,  and  are  usually  distributed  to  the 
blood  vessels. 

Exercise  No.  41. — Harden  the  spleen;  mount,  stain 
and  examine.  Follow  the  formalin  method;  stain 
with  eosin  and  haematoxylin. 

Exercise  No.  42. — Do  the  same  with  a  lymph  nodule. 
THE  THYMUS. 

The  thymus  is  a  gland.  In  early  life  it  is  mostly 
ephithelial  in  nature,  and  in  later  life  it  becomes  a 
lymphoid  gland,  presenting  all  the  characteristics  of 
the  spleen.  The  blood  vessels  and  nerves  are  found 
in  the  trabeculae  and  send  other  branches  into  the 


HISTOLOGY.  69 

lymphoid  tissue.  The  thymus  is  the  only  lymphoid 
gland  that  has  no  germ  center,  but  cell  division  can  be 
demonstrated  in  the  gland.  Hassal's  corpuscles  are 
the  dense  bodies  found  in  the  thymus.  They  are  re- 
garded as  epithelial  remains  of  the  gland  in  early  life. 

THE  TONSILS. 

The  tonsils  are  situated  in  the  mouth  at  the  base  of 
the  tongue.  They  are  surrounded  by  a  dense  mem- 
brane of  white  fibrous  tissue  on  the  under  side,  and  by 
the  mucous  membrane,  which  is  made  up  of  pavement 
epithelial  cells  of  the  buccal  cavity,  on  the  outer  side. 
The  membrane  dips  down  sometimes  into  the  sub- 
stance of  the  tonsil,  and  is  called  the  crypts.  The  ton- 
sils are  supplied  with  blood  vessels,  lymphatics .  and 
nerves,  which  enter  the  organ  through  the  capsule. 
They  are  lymphoid  bodies. 

SEROUS  CAVITIES  AND  MEMBRANES. 

The  serous  membranes  may  be  considered  as  ex- 
panded lymphatics.  These  membranes  are  composed 
of  a  thin  sheet  of  fibrous  tissue,  supporting  a  layer  of 
thin,  irregular,  polyhedral  cells,  placed  edge  to  edge 
and  united  by  a  cement  substance.  At  very  short  dis- 
tances there  are  little  openings  in  the  mucous  mem- 
brane, called  stomata,  which  are  the  mouths  of  lym- 


70 


LABORATORY  METHODS   OP 


phafic  g-lands.  Sometimes  the  cells  do  not  adjust 
themselves  completely,  and  a  space  is  left,  called  pseu- 
do-stomata.  The  pleura,  pericardium  and  peritoneum 
are  serous  membranes.  The  serous  membranes  are 
not  in  contact  with  the  air. 

THE  MUCOUS  MEMBRANES. 

This  variety  of  tissue  is  in  such  a  position  that  it 
may  come  in  contact  with  the  air.  It  is  composed  of 
the  following  structures :  epithelial  cells,  basement 


DIAGRAM  OF  THE  DIFFERENT  TYPES  OF  GLANDS. 

1,  Simple  tube;   2,  compound  tube;   3,  simple  coiled  tube; 

4,   compound  saccular;    5,   simple  saccular, 


HISTOLOGY.  71 

membrane,  and  beneath  the  basement  membrane  there 
is  a  layer  of  smooth  muscle,  called  muscularis  mucosie. 
Along  the  course  of  the  membrane  there  are  several 
openings  which  represent  the  months  of  glands. 
These  glands  are  simply  an  infolding  of  the  mucous 
membrane.  Several  types  of  glands  are  recognized. 
All  of  them  are  some  type  of  a  tube.  See  cut  for 
types  of  glands.  The  different  glands  will  be  dis- 
cussed as  they  are  studied. 


CHAPTER  XL 


THE  NERVES. 


The  nerves  are  highly  specialized  fibers,  beginning  in 
a  cell  and  ending  in  a  special  end-organ.  Their  func- 
tion is  to  carry  impulses  to  and  from  the  cell.  The 
combination  oi  a  cell,  end-organ,  and  nerve  fiber,  is 
called  a  neuron.  A  nerve  cell  is  one  of  the  largest  cells 
in  the  body  and  possesses  all  the  essentials  of  any 
cell,  viz. :  nucleus,  nucleolus  and  protoplasm.  They 
have  no  cell  wall.  The  cells  vary  greatly  in  shape,  and 
each  point  of  the  cell  is  called  a  pole.  One  of  the  points 
is  called  an  axis  cylinder.  The  points  arising  from  the 
cell  at  other  points  than  the  one  called  axis-cylinder 
are  called  dendrites.  The  nerves  are  named  according 
to  the  number  of  processes  they  have.  The  first  type 
and  second  type  cells  are  those  cells  that  have  either  a 
long  or  short  axis  cylinder.  The  first  is  a  first  type 
cell,  and  the  second  is  a  second  type  cell.  Neurogha 
cells  are  small  cells  that  give  off  several  short  proc- 
esses, which  act  as  a  connective  tissue.  The  cell  is 
called  a  glia  cell,  and  the  fibrils  are  called  neuroglia. 
(72) 


HISTOLOGY.  73 

GANGLIA. 

These  bodies  are  nerve  centers,  consisting  of  nerve 
cells  and  nerve  fibers.  Some  of  the  fibers  end  in'  the 
ganglion,  while  others  pass  on  to  more  distant  points. 
The  brain  and  spinal  cord  may  be  termed  a  group  of 
large  ganglia.  The  whole  ganglion  is  covered  with  a 
fibrous  tissue,  called  a  sheath. 

NERVE 


The  nerve  fiber  is  composed  of  the  axis  cylinder  of  a 
nerve  cell,  and  three  protecting  membranes:  the  axi- 
lemma,  the  medullary  sheath  and  the  neurilemma. 

In  the  medullary  nerves,  at  regular  intervals,  there 
appear  constrictions,  called  the  nodes  of  Ranvier. 
These  nodes  are  the  result  of  an  absence  of  the  medul- 
lary sheath  at  these  points.  The  space  between  any 
two  nodes  is  called  internode.  In  the  center  of  an  in- 
ternode  is  found  the  nerve  corpuscle,  located  directly 
under  the  neurilemma.  The  non-medullated  nerve 
fibers  are  found  in  the  sympathetic  system.  Each  fiber 
consists  of  an  axis  cylinder,  neurilemma  and  an  oval 
nucleus  upon  the  surface  of  the  fiber.  There  are  two 
theories  concerning  the  formation  of  the  axis  cylinder  : 
one  is  that  the  nerve  cells  fuse  themselves  together, 
end  to  end,  ancj  form  a  long  thread;  the  other  is  that 


74  LABORATORY  METHODS  OF 

the  nerve  cells  have  a  large  number  of  processes,  and 
one  of  these  functions  as  an  axis  cylinder.  In  both 
cases  the  neurilemma  corresponds  to  a  cell  membrane. 
A  medullated  fiber  is  a  fiber  with  a  medullary  sheath, 
and  a  non-medullated  fiber  is  a  fiber  without  a  medul- 


T.  S.  SPINAL  CORD. 

Showing  investing  membranes   and   distribution  of  blood 
vessels  in  the  white  and  gray  substances. 

lary  sheath.  The  nerves  end  in  the  muscle  tissue  by 
expanding  into  a  small  plate  of  nerve  fiber.  There  are 
two  main  divisions  of  nerves,  viz. :  central  and  sympa- 
thetic nerves.  We  will  study  each  of  these  in  detail, 


HISTOLOGY.  75 

The  central  nerves  are  studied  by  taking  a  section 
of  the  spinal  cord  and  "dissociating  the  tissue,  then 
scraping  the  anterior  cornua  and  spreading  the  ma- 
terial on  a  slide  and  examining  it  in  the  fresh  state. 
The  best  method  is  to  study  a  stained  section  and  com- 
pare results  with  the  cuts  in  all  good  books  on  the 
subject. 

Exercise  No.  43. — Take  a  T.  $.  of  the  spinal  cord  of 
a  dog  and  scrape  the  anterior  cornua,  and  examine 
the  scrapings. 

Use  the  saline  rnount;  search  for  the  following:  A 
large  nerve  cell  having  its  processes.  Locate  the  axis- 
cylinder  of  the  cell. 

Exercise  No.  44. — Examine  the  nerve  of  a  dog  or  ox 
in  salt  solution. 

Technic  same  as  above. 

Exercise  No.  45 — Make  a  T.  S.  of  spinal  cord  from 
tissue  that  has  been  hardened.  Stain,  mount  and 
preserve. 

Search  for  the  following:  the  gray  matter,  consist- 
ing of  ganglion  cells,  neuroglia  and  fibers ;  the  white 
matter,  consisting  of  meduHated  fibers  and  connective 
tissue.  'The  white  matter  surrounds  the  gray.  The  gray 


76  LABORATORY  METHODS  OF 


T.  S.  SPINAL,  CORD— CAT. 

1,  Anterior  median  fissure;  2,  posterior  median  fissure; 
3,  anterior  roots;  4,  posterior  roots;  5,  central  canal;  6, 
anterior  gray  commissure;  7,  posterior  gray  commissure; 
8,  anterior  white  commissure;  9,  posterior  white  commis- 
sure; 10,  anterior  columns;  11,  posterior  lateral  column; 
12,  posterior  column;  13,  substantia  gelatinosa;  14,  15,  16, 
17,  18,  groups  of  motor  cells;  19,  column  of  Burdoch;  20, 
column  of  Goll. 

matter  is  in  the  shape  of  the  letter  H.  The  surface  of 
the  cord  is  divided  into  four  areas:  anterior,  pos- 
terior and  two  lateral.  On  dividing  the  cord  into 
halves,  from  before  backwarc)s?  and  then  dividing 


HISTOLOGY.  77 

each  half  into  three  columns,  we  have  the  anterior, 
posterior  and  lateral  columns  of  each  half,  making 
six  columns  in  the  cord.  In  the  lower  cervical  and  up- 
per thoracic  region  two  more  divisions  appear  in  the 
posterior  column,  the  column  of  Goll  (placed  near  the 
posterior  fissure)  and  the  column  of  Burdoch.  The 
white  matter  is  connective  tissue  and  nerve  fibers,  hav- 
ing a  large  number  of  glia  cells  interspersed  in  them. 
The  nerve  fibers  have  a  medullary  sheath,  but  no  neit- 
rilemma.  "The  nerve  fibers  run  in  the  same  direction 
as  the  long  axis  of  the  spinal  cord ;  a  few  may  be  seen 
which  run  across  the  cord." — Leroy.  Around  the  en- 
tire cord  are  found  three  protecting  membranes:  the 
dura  mater,  arachnoid  and  pia  mater.  The  space  be- 
tween the  dura  mater  and  arachnoid  is  called  the  sub- 
dural  space.  The  space  between  the  arachnoid  and 
pia  mater  is  called  the  sub-arachnoidaj  space.  All 
these  membranes  are  composed  of  white  fibrous  tissue 
and  endothelial  cells.  They  are  all  more  or  less  con- 
nected by  fine  fibers  of  fibrous  tissue,  which  run  trans- 
versely to  the  long  axis  of  the  cord.  The  Pacchion- 
ian  bodies  are  large  papillae  projecting  upward  into  the 
subdural  space.  The  blood  vessels  are  found  in  the 
dura  mater  and  pia  mater;  but  none  are  found  in  the 
arachnoid.  They  are  most  numerous  in  the  pia  mater. 
The  gray  matter  of  the  cord  is  arranged  in  the  shape 
of  the  letter  H.  The  long  arms  of  the  H  are  directed 


78  LABORATORY  METHODS  OF 

from  before  backwards,  and  the  short  arm  is  directed 
from  side  to  side,  joining  the  long  arms  at  their  mid- 
dle points.  The  long  arms  are  comma-shaped,  with 
the  big  end  of  the  comma  directed  anteriorly,  i.  e.,  to- 
wards the  front  of  the  cord.  This  end  does  not  ap- 
proach the  surface  as  near  as  the  small  end  of  the 
comma.  The  small  end  is  called  the  posterior  horn, 
while  the  large  end  is  called  the  anterior  horn  of  the 
cord.  In  the  center  of  the  transverse  arm  of  the  H.  is 
seen  a  small  opening,  called  central  canal,  lined  with 
ciliated  epithelial  cells,  and  containing  a  fluid  called  cer- 
ebro  spinal  fluid.  The  transverse  arm  of  the  H  is  called 
the  gray  commissure,  and  the  white  matter  imme- 
diately in  front  and  behind  it  is  called  respectively  an- 
terior and  posterior  white  commissure.  Some  histolo- 
gists  divide  the  gray  commissure  into  anterior  and 
posterior,  with  reference  to  central  canal.  There  is  a 
narrow  cleft  in  the  white  substance,  beginning  at  the 
anterior  median  periphery,  and  extending  to  within  a 
short  distance  of  the  gray  commissure,  called  anterior 
median  fissure.  A  narrower  but  similar  fissure  is 
seen  on  the  posterior  side  of  the  gray  commissure, 
called  posterior  median  fissure.  On  each  limb  of 
the  letter  H  of  the  cord  there  are  three  horns;  an- 
terior, posterior  and  lateral  horns,  located  as  their 
names  indicate.  From  the  anterior  horn  emerge  the 
anterior  roots,  and  the  posterior  horns  receive  the  pos- 


HISTOLOGY.  79 

terior  roots.  From  the  lateral  horn  emerge  the  motor 
nerve  axis-cylinders,  the  same  as  in  the  anterior  horn. 
The  nerve  cells  of  the  anterior  horn  resemble  a  -straight- 
ened comma,  with  a  very  long  tail.  The  reticular 
processes,  column  of  Clark,  and  the  substantia  gelatin- 
osa,  enter  the  structure  of  the  posterior  horn.  The 
substantia  gelatinosa  covers  the  horn  and  surrounds 
the  central  canal.  The  nerves  of  the  anterior  horn  and 
the  column  o>f  Clark  are  very  large  and  stellate.  The 
blood  vessels  of  the  cord  enter  it  mostly  from  the  pia 
mater,  and  branch  repeatedly  to  form  capillaries,  which 
run  parallel  with  the  fibers  in  the  wihite  matter,  but 
form  a  net  work  in  the  gray  matter.  In  both  cases, 
they  are  surrounded  by  neuroglia  tissue.  There  are 
thirty-one  pairs  of  nerves  springing  from  the  spinal 
cord.  The  motor  nerves  are  anterior,  while  the  sen- 
sory nerves  are  posterior.  They  are  distributed  to  the 
skin  and  other  parts  of  the  body,  and  terminate  in  a 
special  nerve-ending. 

Exercise  No.  46. — Make  a  T.  $.  of  the  spinal  cord. 

Tcchnic:  Mullers  fluid,  paraffine,  haematoxylon  and 
eosin. 

Locate  the  following:  (L.  P.)  Enclosing  mem- 
branes, median  fissures,  gray  matter,  white  matter, 
axis-cylinders  in  the  white  matter,  T.  S.  with  their  en- 


80  LABORATORY  METHODS  OF 

closing  membranes,  the  horns  of  the  cord  in  the  gray 
matter,  the  different  .shaped  and  sized  nerve  cells  in  the 
anterior  horn,  central  canal,  anterior  and  posterior 
roots,  the  different  commissures,  and  the  entrance  of 
the  blood  vessels  in  the  pia  mater. 

CEREBRUM. 

The  cerebrum  is  composed  of  five  layers  of  nerve 
cells,  which  merge  imperceptibly  into  each  other. 
They  are : 

1.  The  molecular  layer.     This  is  the  first  layer,  and 
is  composed  of  a  number  of  finely  granular  cells,  with 
medullated  fibers  and  neuroglia.     The  cells  of  Cajal 
are  found  in  this  layer. 

2.  l^he  layer  of  small  pyramidal  cells.  This  is  located 
under  the  first  layer  and  the  processes  that  leave  the 
cells  form  a  net  work.     The  axis-cylinders  leave  the 
basal  ends  of  the  cells  and  send  collaterals  in  the  me- 
dulla.    The  dendrites  enter  the  molecular  layer. 

3.  The  layer  of  large  pyramidal  cells.     This  differs 
from  the  second  only  in  size  of  the  cells,  and  in  this 
layer  there  is  no  network  sent  to  the  layer  above. 

4.  The  layer  of  irregular  cells.     This  layer  is  com- 
posed of  oval  or  polygonal  cells,  which  have  no  special 
dendrites.     Each   axis-cylinder   sends    out   collaterals, 
and  enters  the  medulla  to  become  one  or  two  nerve 
fibers. 


HISTOLOGY. 


81 


SECTION  OF  CEREBRUM. 
Showing  morphology  and  arrangement  of  the  cells. 

5.  Spindle-shaped  cells.  This  layer  lies  next  to  the 
medulla,  and  is  composed  of  spindle-shaped  cells,  with 
nerve  fibers  between  them.  The  cells  are  arranged 
parallel  with  the  fibers.  These  five  layers  constitute 
the  cortex  of  the  cerebrum.  In  these,  that  part  of  the 
nervous  force  dedicated  to  the  cerebrum,  is  carried  on. 
The  fibers  extending  from  the  cortex,  with  the  connect- 
ive tissue  structure  holding  the  nerve  fibers  together, 
constitute  the  medulla  of  the  cerebrum. 
6 


82  LABORATORY  METHODS  OF 

The  five  layers  of  the  cerebrum  are  the  molecular 
layer,  the  small  pyramidal  layer,  the  large  pyramidal 
layer,  the  layer  of  irregular  cells  and  the  layer  of  spin- 
dle-shaped cells. 
Exercise  N~o.  47. — Make  a  T.  S.  of  the  cerebrum. 

Technic :  same  as  the  spinal  cord. 

1.  Search  for  the  layer  of  the  granular  cells,  large 
and  small  pyramidal  cells,  and  the  layer  of  irregular 
and  oval  cells. 

2.  Examine  the  medulla  of  the  cerebrum ;  note  the 
presence  of  connective  tissue  cells,  or  glia  cells. 

3.  Search  for  spider  and  Cajal  cells.     In  the  first 
layer  will  be  found  the  cells  of  Cajal;  in  the  second 
layer  will  be  found  the  small  pyramidal  cells;  in  the 
third  layer  will  be  found  the  large  pyramidal  cells; 
in  the  fourth  layer  will  be  found  the  irregular  cells; 
in  the  fifth  layer  will  be  found  the  oval  cells. 

CEREBELLUM. 

The  cerebellum  is  composed  of  three  layers:  The 
outer  molecular,  the  middle  granular  or  rust  colored 
layer,  and  the  inner  medullary  tract.  The  outer  layer 
consists  of  three  varieties  of  nerve  cells:  the  cells  of 
Purkinje,  the  polypo-lar  or  multipolar,  and  the  stellate 
cells.  The  middle  layer  contains  two  varieties  of  cells : 
the  granular  cells  and  the  short  stellate  cells.  The 
inner  layer  contains  two  varieties  of  cells :  the  centrifu- 


HISTOLOGY.  83 

gal  neuraxis  of  the  Purkinje  cell  and  the  two  types  of 
centripetal  cells,  one  type  sending  their  neuraxis  into 
the  granular  layer.  These  fibers  are  called  mossy  fibers 
and  do  not  extend  through  the  granular  layer.  The 
other  type  of  cells  sends  the  collaterals  of  the  cells  into 
the  layer  where  the  Purkinje  cells  are  found  and  seem 
to  ascend  the  neuraxis  of  the  Purkinje  cells,  and  are 
called  climbing  fibers  of  the  centripetal  cells.  The 
central  gray  nucleus  are  found  in  the  medullary  por- 
tion of  the  cerebellum,  and  are  a  number  of  multipolar 
cells  with  many  dendrites.  The  medullary  or  white 
substance  of  the  cerebellum  is  arranged  in  a  layer  of 
branching  sheets,  and  when  cut,  presents  in  the  cut 
section  a  tree-like  arrangement,  called  arbor  vitae.  For 
more  extensive  work  on  this  subject  see  Leroy,  So- 
botta,  and  Bohm  and  Davidoff. 

THE    MEDULLA   OBLONGATA. 

This  structure  is  the  same  as  that  of  the  spinal  cord, 
the  chief  difference  being  in  the  arrangement  of  the 

structures. 

SYSTEM     OF    SYMPATHETIC     NERVES. 

This  system  is  composed  of  a  large  number  of  gan- 
glia and  nerve  fibers,  connected  by  small  fibers.  The 
fibers  are  non-medullated.  They  are  arranged  into 


84  HISTOLOGY. 

three  large  groups,  viz.:  the  cardiac  ganglia,  the  epi- 
gastric ganglia  and  the  hypogastric  ganglia.  These 
ganglia  are  located  in  front  of  the  spinal  column. 

NERVE  ENDINGS. 

There  are  three  varieties  of  nerve  endings :  free 
nerve  endings  occurring  in  the  skin,  mouth,  spinal 
cord,  etc.,  terminal  corpuscles  occurring  in  the  skin, 
and  neuro-epithelial,  occurring  in  the  perceptive  or- 
gans, viz. :  mouth,  eye,  nose  and  ear. 

Exercise  No.  48. — Examine  the  skin  T.  S. 

Technic :  Harden  in  alcohol,  embed  in  celloidin,  and 
stain  with  haematoxylin  and  eosin. 

I.  Search  for  the  four  layers  of  the  cutis-notha,  hair 
follicle,  hair  in  place,  hair  muscle,  sweat  gland,  se- 
baceous gland,  nerve  endings  and  pigment  of  the  skin. 

In  what  layers  are  all  these  objects  found? 

A  sweat  gland  is  a  simple  tubular  gland. 

What  lesson  do  you  get  from  the  skin  bearing  on  the 
nervous  system? 


CHAPTER  XIL 


THE  SKIN  AND  ITS  APPENDAGES. 


The  cutaneous  system  comprises  the  skin,  hair  and 
glands.  The  teeth  are  sometimes  considered  as  be- 
longing to  this  system.  The  skin  is  composed  of  two 
distinct  structures,  viz. :  the  cuticle  and  cutis.  The 
cuticle  is  derived  from  the  ectoderm  and  the  cutis  is 
derived  from  the  mesoderm.  The  cuticle  consists  of 
four  layers  ;  the  stratum  corneum,  the  stratum  lucidum, 
the  stratum  granulosum  and  the  stratum  Malpighii. 
The  first  layer  is  composed  of  cells  that  have  lost  the 
appearance  of  ever  having  been  nucleated,  called  the 
cornified  layer.  The  second  layer  is  composed  of  cells, 
wilier!  are  polyhedral  in  shape  and  have  almost  lost 
their  nuclei.  The  third  layer  is  composed  of  cells  ir- 
regularly polyhedral  in  shape  and  are  joined  together 
by  little  spines.  These  cells  are  called  prickle  cells, 
and  outside  of  these  are  seen  other  cells,  which  have  a 
granular  protoplasm.  These  two  layers  constitute  the 
stratum  granulosum.  The  fourth  layer  is  composed 
of  cells,  which  are  columnar  in  shape  and  are  in  a 
stage  of  active  growth,  as  shown  by  the  property  they 

(85) 


86  LABORATORY  METHODS  OF 


L.  S.   SKIN.— HUMAN. 

1,  stratum  corneum;  2,  stratum  lucidum;  3,  stratum 
granulosum;  4,  stratum  •Malpighii;  5,  corium;  6  and  7, 
vessels  of  the  skin. 

have  of  straining  so  easily.  This  layer  is  the  one  that 
contains  the  pigment  that  gives  color  to  the  skin  in  the 
colored  races  and,  in  some  locations,  in  the  white  race. 
This  layer  rests  upon  the  true  skin.  The  true  skin  is 
composed  of  two  layers  of  fibro-elastic  tissue,  called 
papillae  of  the  true  skin.  The  nerves  end  in  the  true 
skin,  in  end  corpuscles,  and  in  the  cuticle  in  an  arboriz- 
tioti  manner. 


HISTOLOGY.  87 

TH£  SEBACEOUS  GLANDS. 

These  glands  are  of  the  compound  saccular  type  and 
are  located  in  the  papillae  of  the  corium.  They  consist 
of  acini,  secreting  cells  and  a  duct.  The  sudoriferous 
glands  belong  to  the  tubular  type,  and  consist  of  a 
basement  membrane  and  gland  cells  and  a  duct.  The 
gland  is  located  deep  down  in  the  tissue  beneath  the 
skin.  The  deep  end  is  coiled  upon  itself  and  the  su- 
perficial end  is  straight  nearly  all  the  way  from  the 
coil  to  the  cuticle.  Just  at  the  cuticle  the  duct  becomes 
tortuous  and  makes  its  way  out  as  a  little  depression  on 
the  surface  of  the  skin.  All  these  structures  will  be 
seen  when  you  study  the  skin. 

NAILS  AND  HAIR. 

The  nails  are  derived  from  the  epidermis,  and  are 
composed  of  horny  cells.  The  parts  of  a  nail  are 
nail-body,  nail-bed  and  nail-matrix.  The  nail-bed  is 
composed  of  the  stratum  Malpighii.  The  nail,  there- 
fore, grows  in  length  and  thickness  by  new  cells  from 
the  matrix.  The  body  of  the  nail  is  composed  of  the 
other  layers  of  the  cuticle.  The  stratum  lucidum  takes 
the  greater  part  in  the  formation  of  the  nail  as  the 
transparency  indicates. 

The  hair,  like  the  nail,  is  derived  from  the  skin,  and 
is  found  almost  all  over  the  body.  There  are  no  hairs 


88  LABORATORY  METHODS  OF 

in  the  palms  of  the  hands  and  soles  of  the  feet.  The 
hair  consists  of  three  divisions:  the  shaft,  that  part 
above  the  skin;  the  root,  that  part  in  the  skin;  the 
bulb,  that  part  from  which  the  hair  grows — in  fact, 
it  is  a  part  of  the  root.  The  hole  in  which  the  hair 
rests  is  called  the  follicle.  The  shaft  consists  of  three 
layers :  the  corticle,  cortical  and  medulla.  The  cuti- 
cle is  the  outermost  covering  and  consists  of  epithelial 


L.    S.    HUMAN    HAIR. 

1,  cuticle;  2,  inner  root  sheath;  3,  Henle's  layer;  4,  me- 
dulla of  hair;  5,  papillae  of  hair;  6,  hair  bulb;  7,  vessels  of 
hair. 


HISTOLOGY.  89 

cells  overlapping  each  other.  The  cortical  is  the  thick- 
est and  contains  the  coloring-  matter  of  the  hair.  The 
medulla  is  not  always  present,  but  when  it  is,  it  con- 
tains air  and  gives  an  appearance  according  to  tta 
light  used  to  examine  the  specimen. 

At  the  bottom  of  the  hair  follicle  is  the  fibrous  tissue 
that  contains  the  blood  vessels  of  the  hair.  The  sheaths 
of  the  hair  are  named  according  to  their  location.  The 
arrectores  muscles  of  the  hair  are  composed  of  a  few 
fibers  of  smooth  muscle-tissue  arising  in  the  fibrous 
of  the  corium,  and  cause  the  hair  to  stand  on  ends  in 
case  of  fright.  A  longitudinal  section  of  the  hair  pre- 
sents for  examination :  shaft,  composed  of  three  lay- 
ers of  modified  epithelial  cells ;  root,  that  part  of  the 
hair  within  the  skin;  the  bulb,  a  division  of  the  root; 
the  papillae,  an  elevation  of  the  true  skin  into  the  root 
of  the  hair;  the  sheaths,  surrounding  the  root  and 
bulb,  called  root  sheaths  (the  opening  and  the  sheaths 
are  called  the  hair  follicle)  ;  sebaceous  glands,  com- 
posed of  a  saccular  variety  of  glands,  located  about 
midway  the  root  of  the  hair. 

The  theories  as  to  the  growth  of  hair  are  two :  first, 
that  the  hair  grows  from  an  infolding  of  the  epidermis 
of  the  skin ;  second,  that  the  hair  grows  from  the  papil- 
lae at  the  bottom  of  the  root.  The  nerves  of  the  hair 
are  few  and  extend  only  to  the  duct  of  the  sebaceous 


90  LABORATORY  METHODS   OP 

gland.  There  is  only  one  fiber  to  each  hair.  The 
shedding  -of  the  hair  is  a  difficult  subject  to  under- 
stand, but  the  theory  is  that  the  cells  in  the  papillae 
harden  and  therefore  the  hair  loosens  and  falls  out. 
These  are  called  bulb  hairs.  The  papillary  hairs  are 
shed  in  this  way  from  the  external  root  sheath  there 
arises  a  bud,  which  grows  downward  and  develops 
into  a  new  hair,  which  gradually  pushes  the  old  hair 
out.  The  condition  called  goose  flesh  is  caused  by  a 
contraction  of  the  muscles  of  the  hair.  The  nerves 
supplying  the  muscles  of  the  hair  are  derived  from  the 
sympathetic  nerves  and  are  called  pilomotor  nerves. 

TH£  MAMMARY  GLANDS. 

These  glands  are  included  in  the  cutaneous  glands. 
They  are  a  variety  of  compound  saccular  glands,  and 
are  histologically  considered  as  a  tube  being  lined  with 
a  basement  membrane  and  glandular  epithelial  cells 
with  large  nuclei.  The  opening  in  the  gland  is  called 
acinus.  The  end  of  the  tube  is  called  the  duct.  A 
large  number  of  these  glands  is  put  together  and  forms 
a  lobule  having  a  small  amount  of  white  fibrous  and 
connective  tissue  placed  around  it.  A  large  num- 
ber of  these  lobules  is  put  together  and  forms  a  lobe. 
The  gland,  as  we  see  it,  is  a  number  of  glands — i.  e., 
just  as  the  liver  is  composed  of  separate  livers,  so  is 


HISTOLOGY. 


91 


T.   S.  MAMMARY  GLAND. 

Showing  lobules,  connective  tissue,  and  acini  lined  with 
columnar  epithelial  cells. 

this  gland.  The  mammary  is  richly  supplied  with 
blood  vessels  and  lymphatics.  The  lymphatices  of 
this  gland  communicate  with  those  of  the  axilla.  The 
secretion  of  it  is  called  milk.  Milk  is  a  mixture  of 
fat,  leukocytes  and  colostrum  corpuscles. 

Exercise  No.  49. — Make  a    T.    S.    of   the   mammary 
gland. 

Technic:     Formalin,  celloidin  and  H.  &  E. 
Look  for  the  lobe,  lobule,  acinus  and  connective  tis- 
sue elements. 


CHAPTER  XIIL 


THE  DIGESTIVE  TRACT. 


THE;  MOUTH. 

The  mouth  is  lined  with  a  mucous  membrane  con- 
sisting of  stratified  epithelial  cells.  Upon  the  surface 
of  the  mouth  are  seen  the  ducts  of  numerous  glands. 
Between  this  layer  of  epithelial  cells  and  the  submu- 
cous  tissue  are  found  the  papillse  of  the  mouth.  They 
are  composed  of  fibrous  tissue.  The  gums  have  no 
glands  in  them.  The  blood  supply  is  derived  from  the 
mucous  membrane  blood  supply.  The  lymphatics  are 
from  the  same  source. 


THE: 


The  teeth  are  said  by  some  to  be  a  part  of  the  tegu- 
mentary  system.  The  structure  of  a  grown  tooth  is  as 
follows  :  every  tooth  has  a  neck,  crown  and  root.  The 
crown  is  composed  of  a  modified  epithelial  cell  tissue, 
called  enamel.  The  enamel  is  the  hardest  substance 
in  the  body,  and  is  covered  by  a  membrane  of  epithelial 
(92) 


HISTOLOGY.  93 

cells,  the  cuticula  dentis.  The  enamel  substance  is  ar- 
ranged in  columns  or  prisms  called  enamel  prisms. 
They  are  stuck  together  with  a  substance  more  dense 
than  themselves,  called  enamel-cement.  The  lines  of 
Retzius  are  parallel  lines  running  from  the  dentine  to 
the  cuticula  dentis  in  the  enamel.  They  represent  the 
periodic  deposition  of  the  salts  of  lime  in  the  enamel. 

THE  DENTINE. 

The  dentine  is  the  tissue  between  the  enamel  and  the 
pulp-cavity.  It  is  composed  of  little  tubes  about  two 
and  one-half  microns  in  diameter.  They  are  surround- 
ed by  the  membrane  of  Newmann.  In  their  course  they 
take  an  S-shaped  direction,  giving  rise  to  the  lines  of 
Schrager.  Peculiarly,  irregularly  branched  spaces  are 
seen  in  the  dentine,  called  interglobular  spaces.  They 
represent  the  uncalcified  spaces  in  the  dentine. 

The  cementum  is  located  on  the  tooth  from  the 
crown  downward,  and  is  composed  of  bone  plates  hav- 
ing no  Haversian  canals  as  a  rule.  The  peculiarity 
of  the  cementum  is,  it  has  a  large  number  of  Sharpey's 
fibers,  which  are  abundant  in  those  arears  where  noe 
bone  corpuscles  are  found.  They  are  found  where  the 
bone  has  not  calcified. — Bohm  and  Davidoff. 

The  pulp  cavity  is  the  cavity  found  in  the  central 
portion  of  the  tooth,  and  contains  the  pulp.  It  is  very 


94- 


LABORATORY  METHODS  OF 


large.  The  pulp  is  composed  of  fibrous  tissue,  nerve 
fibers,  a  semifluid  substance  and  an  artery.  The  ar- 
tery enters  the  cavity  and  breaks  up  into*  capillaries. 
The  nerves  supply  the  tooth  substance  by  the  branch- 
ing method. 

The  epiblast  and  hypoblast,  both  enter  into  the  com- 
position of  the  tooth.  The  epithelial  of  the  primitive 
jaw  becomes  thickened  and  grows  down  into  the  sub- 
jacent tissue  and  forms  the  dental  ridge.  From  the 
lower  outer  border  of  this  flask-shaped  mass  of  cells, 


L.    S.    TOOTH. 

1,   enamel;    2,   dentine;    3,   pulp   cavity;    4,   neck  of  the 
tooth;   5,  cementum;   6,  pericementum ;   7,  apical  foramen. 


HISTOLOGY.  95 

dental  bulbs  grow  (one  for  each  milk  tooth),  and  sub- 
sequently become  hollow  on  the  under  surface  and  cov- 
er a  conoid  upgrowth  of  connective  tissue  cells  (the 
papillae).  The  dental  bulbs  continue  to  expand  one 
for  each  tooth,  assuming  the  form  of  the  crown  of  the 
tooth,  and  the  inner  layer  of  the  cells  become  columnar. 
Each  of  these  columnar  cells  secretes  an  enamel  prism, 
and  the  bulb  is  now  called  the  enamel  organ.  "While 
this  has  been  in  progress  the  outer  cells  of  the  papillae 
have  elongated  to  form  the  odontoblasts  which  deposit 
the  dentin.  From  what  has  been  said,  it  will  be  seen 
that  the  enamel  is  deposited  outwards  and  the  dentin 
inwards.  The  remains  of  the  papillae  become  the  pulp 
of  the  tooth.  The  permanent  teeth  are  formed  simi- 
larly. The  cementum  is  deposited  later  by  the  alveolar 
periosteum." — Lcroy. 

Exercise  No.  50 — Examine  the   L.    $.    of    the    tooth 
supplied. 

Make  out  the  structure  enamel. 
Exercise  No.  51.— Examine  the  T.  S.  of  the  tooth. 

ESOPHAGUS. 

The  esophagus  is  composed  of  four  coats,  viz. :  mu- 
cous, sub  mucous,  muscularis  mucosae,  muscular  and 
fibrous  coat. 


96  '  LABORATORY  METHODS   OF 

The  mucous  coat  is  composed  of  stratified  epithelial 
cells  resting  upon  a  basement  membrane. 

The  muscularis  mucosse  is  composed  of  a  .band  of 
smooth  muscle  located  just  under  the  mucous  coat. 

The  submucous  coat  consists  of  fibrous  tissue,  lym- 
phatics, nerves,  blood  vessels  and  glands,  which  empty 
their  contents  into  the  esophagus. 

The  muscular  coat  is  composed  of  two  layers  of 
muscle  tissue,  the  inner  layer  being  placed  circularly 
and  the  outer  layer  placed  longitudinally.  The  upper 
third  is  composed  of  the  involuntary  variety. 

The  fibrous  coat  is  composed  of  a  thin  layer  of  white 
fibrous  tissue  and  a  few  fibers  of  yellow  elastic  tissue. 

Exercise  No.  52. — Make  a  T.  $.  of  the  esophagus. 

Technic:  Formalin,  celloidin  and  H.  &  E. 
Make  out  all  the  coats  and  describe  the  tissue  in 
each. 

STOMACH. 

The  stomach  consists  of  the  usual  four  coats :  mu- 
cous, submucous,  muscular  and  fibrous.  The  muscu- 
laris mucosae  belongs  to  the  mucous  coat. 

The  mucous  coat  consists  of  a  basement  membrane 
resting  upon  a  stroma  of  connective  tissue,  and  sup- 
porting a  layer  of  columnar  epithelial  cells.  In  this 


HISTOLOGY.  97 

layer  of  cells  are  found  a  few  goblet  cells.  The  glands 
of  the  stomach  are  of  two  varieties;  the  peptic  and 
pyloric.  The  peptic  are  of  the  tubular  variety,  while 
the  pyloric  are  of  the  compound  tubular  variety,  and 
are  located  in  the  pyloric  end  of  the  stomach.  Under 
the  microscope  the  type  of  glands  will  tell  what  end 
of  the  stomach  is  under  consideration.  The  peptic 
glands  secrete  pepsin,  and  the  gastric  glands  secrete 
the  acid  of  the  stomach. 

The  submucous  coat  consists  of  loose  fibrous  tissue, 
lymphatics,  blood  vessels,  nerves  and  occasionally  a 
little  diffuse  adenoid  tissue  is  found.  This  layer  is 
thrown  up  into  folds,  forming  the  rugae  and  depres- 
sions of  the  stomach. 

The  muscular  coat  consists  of  three  coats  of  mus- 
cle tissue  ;  an  inner  circular,  a  middle  longitudinal  and 
an  outer  oblique  layer. 

The  fibrous  coat  is  made  up  of  white  fibrous  tissue 
derived  from  the  peritoneum,  and  is  covered  on  the 
outside  with  endothelial  cells.  The  outer  coat  of  mus- 
cle tissue  is  usually  absent.  The  lymph-follicles  are 
not  numerous  in  the  stomach. 


SMALL  INTESTINES. 

The  small  intestines  consist  of  the  usual  four  coats, 
arranged  in  the  usual  manner.  The  folds  in  the  in- 
testines are  called  villi,  and  are  composed  of  the  same 


7 


98         LABORATORY  METHODS  OF 


T.  S.  SMALL  INTESTINES. 

1,  mucosa,  covered  with  columnar  epithelial  cells;   2,  sub- 
mucosa;  3,  muscular  layers;  4,  fibrous  layer. 

tissues  as  the  rugae  of  the  stomach.  The  goblet  cells 
are  abundant  in  this  tissue.  In  this  coat  there  are  a 
few  smooth  muscle  fibers  which  give  rise  to  the  mus- 
cularis  mucosae.  The  submucous  coat  is  the  same  as 
that  of  the  stomach. 

The  muscular  coat  is  made  up  of  two  layers :  an  in- 
ner circular  layer  and  an  outer  longitudinal  layer, 
separated  by  a  small  amount  of  connective  tissue. 


HISTOLOGY.  99 

The  serous  coat  is  the  same  as  that  of  the  stomach. 


SMALL    INTESTINE.— CAT. 

1,  villus;  2,  submucosa;  3,  muscularis  mucosae;  4,  Bnm- 
ner's  gland  located  in  the  submucosa. 

The  glands  of  the  small  intestines  are  of  four  vari- 
eties :  Lieberkuhn's  glands  are  found  in  the  mucosa 
of  the  entire  intestinal  tract,  between  the  villi.  They 
are  of  the  simple  tubular  type.  The  glands  of  Brunner 
are  of  the  same  type  as  the  ones  above,  but  divide  into 
many  branches;  therefore  they  look  like  a  racemose 
gland.  They  are  serous  and  not  of  mucous  type. 
These  are  the  duodenal  glands.  The  solitary  glands 
are  found  throughout  the  intestinal  tract  and  are  lymph 


\ 
100  LABORATORY  METHODS   OF 


THREE    VILLI,    SHOWING    THE    ARRANGEMENT    OP 
THE    VESSELS. 

follicles.  "Foyer's  patches  are  elongated  structures 
consisting  of  from  ten  to  sixty  .adjacent  solitary 
follicles  connected  by  their  cortical  zones." — Sobotta. 
They  occur  in  the  mucosa  and  submucosa.  The  nerve 
supply  of  the  intestinal  tract  is  derived  from  the  sym- 
pathetic and  cranial  nerves. 

Exercise  No.    53. — Examine   the   fallowing:   esopha- 
gus, stomach,  s\mal!  intestines  and  large  intestines. 

Technic:  Corrosive  sublimate  and  celloidin.     Stain 
with  H.  Car.  and  H.  &  E. 


HISTOLOGY.  101 

THE  LARGE  INTESTINES. 


SECTION   OP  DOG'S  LARGE   INTESTINE.— PREPARED 

BY  A.  L.  PAEY. 

1,  lumen  of  gland;  2,  columnar  epithelial  cells;  3,  gob- 
let cells;  4,  mucosa;  5,  submucosa;  6;  muscular  layers; 
7,  fibrous  layer. 

THE  LARGE  INTESTINES. 

The  large  intestines  differ  only  from  the  small  in 
that  they  have  a  fewer  number  of  glands  in  them.  The 
lymph  follicles  and  the  glands  of  Lieberkuhn  are  the 


102 


LABORATORY  METHODS  OP 


T.    S.    LARGE    INTESTINES    SHOWING    LYMPH    NOD- 
ULES. 

ones  found.  The  rectum  is  lined  with  stratified  epithe- 
lial. Its  mucous  membrane  is  thrown  into  folds  called 
rectal  valves. 

THE  UVER. 

The  liver  is  composed  of  lobes  and  lobules,  having 
varying  amounts  o>f  connective  tissue  between  each  lob- 
ule and  between  each  lobe.  This  fibrous  tissue  is 
called  the  capsule  of  Glisson.  A  lobule  of  the  liver  is 
composed  of  the  following:  beginning,  in  the  center, 
you  find  the  intralobttlar  vein;  surrounding  this  (with 


HISTOLOGY. 


103 


H.  P.),  you  see  the  liver  cells;  following  these  to  the 
outer  edge  of  the  lobule,  you  see  the  interlobular  vein 
enclosed  in  a  few  fibers  of  Glisson's  capsule.  At  the 
same  point  you  see  the  bile  capillaries  leaving  the  lob- 
ule. 

The  blood  of  the  interlobular  branch  of  the  hepatic 
artery  and  the  blood  of  the  interlobular  branch  of  the 
portal  vein  mix  and  pass  into  the  lobule  between  two 
lobules,  called  the  interlobular  vein,  and  then  pass  to 
the  center  and  out  of  the  lobule  through  the  intralobu- 


A—  T.  S.  LIVER— CAT. 
1,  lobules;  2,  interlobular  connective  tissue. 
B. — 1,  liver  cells. 


104  LABORATORY   METHODS   OP 

lar  vein.  A  number  of  these  intralobular  veins  unite 
to  form  the  sublobular  vein,  and  these  again  unite  to 
form  the  hepatic  veins,  which  carry  the  blood  to  the 
inferior  vena  cava.  Between  each  liver  cell  are  seen 
the  beginning  of  the  bile  capillaries,  which  pass  out  to 
the  edge  of  the  lobule  and  empty  into  the  bile  ducts  in 
that  place.  The  bile  ducts  unite  to  form  the  large  bile 
duct.  The  large  duct  passes  on  to  the  duodenum.' 
When  there  is  more  bile  than  wanted  for  the  digestive 
tract,  the  bile  is  backed  into  the  bile  cyst.  This  cyst 
is  called  gall  bladder,  and  is  placed  on  the  under  side 
of  the  liver.  The  bladder  and  ducts  are  composed  of 
white  fibrous  tissue  lined  with  columnar  epithelial  cells. 
Under  this  is  the  muscle  tissue,  and  under  the  muscle 
tissue  is  some  loose  areola  tissue — then  comes  the  layer 
of  fibrous  tissue,  lined  with  endothelial  cells.  The  lym- 
phatics are  in  the  form  of  perivascular  lymphatics. 
The  nerves  are  of  both  varieties :  medullated  and  non- 
medullated.  The  liver  cells  form  the  chief  study  of 
the  liver. 

Exercise  No.  54. — Study  all  the  tissue  from  the  in- 
jected tissue  furnished.  And  make  T.  $.  of  the 
tissue  furnished. 

Stain,  mount,  study  and  preserve. 


HISTOLOGY.  105 

TH£   SALIVARY   GLANDS. 

The  salivary  glands  are  the  parotid,  submaxillary, 
sublingual  and  the  pancreas.  They  are  all  of  a  true 
glandular  nature,  having  a  duct,  which  divides  and 
subdivides  until  it  reaches  the  alveolus  of  the  gland. 
The  little  alveolus  is  in  reality  the  lumen  of  the  duct 
lined  with  glandular  epithelial  cells.  Kach  individual 
alveolus  and  its  basement  membrane  is  called  a  lobule. 
Each  lobule  has  an  enclosing  membrane  of  white  fi- 
brous tissue  separating  it  from  the  next  lobule.  As 
these  duct  branches  grow  larger  a  second  band  of 
white  fibrous  tissue  is  thrown  around  a  number  of 
them,  forming  a  lobe.  A  band  of  white  fibrous  tissue 
is  thrown  around  the  whole  mass.  This  band  is  called 
a  capsule.  The  whole  mass  is  called  a  gland.  The 
duct,  after  leaving  the  alveolus,  is  lined  with  columnar 
epithelial  cells.  In  some  glands  it  is  lined  with  two 
layers  of  cells.  The  salivary  glands  are  named  ac- 
cording to  their  secretion:  mucous,  serous  and  mixed 
glands.  The  glands  are  richly  supplied  with  blood  ves- 
sels. The  artery  enters  the  gland  at  the  point  where 
the  duct  leaves  it,  and  when  it  reaches  the  acini  it 
breaks  up  into  a  network  of  capillaries  that  reunite  to 
form  the  veins,  which  leave  the  gland,  accompanied  by 
the  artery.  The  lymphatics  are  found  with  the  artery 
as  far  as  the  'acini.  The  nerves  are  of  both  varieties  and 


106  LABORATORY   METHODS   OF 

reach   as   far  as  the  acini.       There  are   found   small 
ganglia  in  the  glands. 

THE  PAROTID  GLAND. 

This  gland  is  a  serous  gland.     It  presents  for  an  ex- 
amination a  capsule,  septa  of  connective  tissue,  lobes 


T.  S.  SUBMAXILLARY  GLAND. 
1,  fibrous   connective  tissue  between  the  lobules. 

and  lobules.  The  lobe  is  composed  of  lobules,  connec- 
tive tissue  between  the  lobules  and  a  duct.  The  lobule 
contains  acini  and  ductules.  The  acini  is  the  terminal 
of  the  tube  of  which  the  gland  is  composed.  The  com- 


HISTOLOGY.  107 

position  of  acini :  glandular  cells,  resting  on  a  base- 
ment membrane  and  the  lumen  of  the  tube,  or  gland. 
The  ductules  discharge  their  contents  into  the  ducts. 

Exercise  No.  55. — Study,  mount  and  preserve  the  sec- 
tion suppliea. 

Note  the  method  of  stain  used  in  your  book. 

l'..rercise  No.  56. — M\a\ke  a  study  of  the  pancreas  in  the 
same  way  you  did  the  parotid. 

Note  the  difference,  if  there  is  any.  The  ducts  of 
all  glands  are  lined  with  columnar  epithelial  cells. 
Sometimes  the  cells  are  arranged  in  two  layers.  (See 
the  scheme  ot  the  salivary-glands  in  Bohm  and  David- 
off,  page  227.) 

Exercise  No.  57. — Make  a  study  of  the  gland  tissue 
supplied  and  make  out  all  the  parts  named  as  be- 
longing to  a  gland. 

Mount  and  preserve. 

Exercise  No.  58. — Make  a  T.  $.  of  all  the  glands  of 
the  month  and  note  the  difference,  if  any,  in  them. 

Make  drawings  of  all  of  them.  There  are  numerous 
small  glands  in  the  lips  and  oral  cavity  generally,  and 
are  named  according  to  their  location. 


108  HISTOLOGY. 

Exercise  No.  59. — Make  T.  $.  of  the  lip. 

Study,  mount  and  preserve. 

THE  PANCREAS. 
The  pancreas  is  the  abdominal  salivary  gland  and  is 


SECTION  OF  PANCREAS. 

in  structure  the  same  as  the  other  glands  of  the  same 
name. 

Exercise  No.  60. — Make  a  T.  $.  of  the  pancreas. 

Study,  mount  and  preserve. 
Note  the  technic  in  all  cases. 


CHAPTER  XIV. 


THE  URINARY  TRACT. 


THE    KIDNEY. 

This  organ  is  a  compound  tubular  gland,  consisting 
of  about  twenty  lobules,  surrounded  by  a  dense  layer 
of  white  fibrous  tissue  called  a  capsule.  In  this  cap- 
sule is  found  some  smooth  muscle  tissue.  The  kid- 
ney is  of  a  bean  shape.  And  the  hilum  is  directed  to- 
wards the  median  line  of.  the,  abdominal  cavity.  At 
the  hilum  the  artery  enters,  and  the  vein  and  ureters 
leave.  The  nerves  are  of  both  kinds :  the  medullated 
and  non-medullated. 

The  outer  investment  is  called  a  capsule,  having  in 
it  a  few  smooth  muscle  fibers.  The  cortex  of  the  kid- 
ney is  located  just  under  the  capsule  and  constitutes 
one-third  of  the  kidney  substance.  It  contains  the 
labyrinth,  Malpighian  bodies  and  uriniferous  tubules. 
The  labyrinth  is .  the  space  found  between  the  upper 
end  of  the  uriniferous  tubules,  and  extends  down- 
wards to  the  point  where  they  all  unite  to  empty  into 

(109) 


HO  LABORATORY  METHODS   OF 

the  calyces  of  the  kidney.  It  will  be  remembered  that 
the  uriniferous  tubules  are  gathered  into  bundles  and 
then  proceed  downwards  until  they  reach  a  point  just 
above  the  calyces,  where  they  join  several  other  bun- 
dles forming  a  mass  of  tubules  which  empty  into  the 
calyces.  There  are  several  such  masses  in  one  kidney. 
These  masses  are  called  the  medullary  substance  of 
the  kidney.  The  beginning  of  the  medullary  substance 
is  nearly  like  the  fingers  of  your  hand.  Hold  your 
hand  up  before  you  and  both  extend  and  separate  your 
fingers.  You  will  then  have  a  fair  example  of  the 
medullary  rays,  medullary  substance,  labyrinth  and 
tube  of  Bellini,  the  fingers  representing  the  medullary 
rays ;  the  spaces  between  them  representing  the  laby- 
rinth, the  palms  representing  the  medullary  substance 
and  the  wrist  the  tube  of  Bellini.  The  tube  of  Bellini 
is  the  excretory  duct  of  the  uriniferous  tubules. 

The  blood  vessels  enter  the  kidney  at  the  hilum  and 
pass  upwards  and  give  off  branches  to  form  the  glorri- 
erulus.  The  glomerulus  is  a  coil  of  an  artery,  which 
enters  the  capsule  of  Bowman  at  the  same  point  where 
the  vein  leaves  the  capsule.  In  this  body  the  urine  is 
separated,  in  part,  from  the  blood.  (For  the  theory  of 
the  secretion  of  urine,  see  the  text  on  Urine.)  The  uri- 
niferous tubule  is  the  tube  which  carries  the  urine  from 
the  glomerulus  to  the  calyces  of  the  kidney,  and  it 


HISTOLOGY. 


Ill 


SCHEME    OF  URINIFEROUS    TUBULES   AND   GLOM- 
ERULI. 

],  afferent  artery;  2,  efferent  vein;  3,  glomerulus;  4, 
neck  of  the  uriniferous  tubule;  5,  proximal  convoluted  tu- 
bule; 6,  spiral  tubule;  7,  descending  limb  of  Henle's  loop; 
8,  Henle's  loop;  9,  ascending  limb  of  Henle's  loop; 
10,  spiral  portion  of  the  ascending  limb;  11,  zigzag  tubule; 
12,  distal  convoluted  tubule;  13,  straight  collecting  tubule. 

leaves  the  glomerulus  at  the  opposite  pole  from  the 
artery  and  vein.  The  uriniferous  tubule  takes  a  tortu- 
ous course  and  has  received  names  accordingly:  I,  the 
neck,  lined  with  low  cuboidal  epithelial  cells;  2,  the 
convoluted  tubule,  lined .  with  low  columnar  cells ;  3, 
the  spiral  tubule,  lined  with  the  same  as  No.  2;  4,  the 
descending  limb  of  Henle,  lined  with  squamous  cells; 


112 


LABORATORY  METHODS  OF 


DIAGRAM   OP  KIDNEY. 

1,  intralobular  artery;  2,  glomerulus;  3,  afferent  vessel 
(vein)  5,  intralobular  vein;  6,  venous  capillaries  of  the 
cortex;  straight  uriniferous  tubule. 

5,  Henle's  loop,  lined  with  squamous  cells ;  6,  the  as- 
cending limb  of  Henle,  lined  with  the  same  sort  of  cells 
as  the  loop;  7,  the  irregular  tubule,  lined  with  striated 
columnar  epithelial  cells;  8,  the  distal  convoluted  tu- 
bule, lined  with  granular  epithelial  cells;  9  the  arched 
collecting  tubule,  lined  with  low  cuboidal  cells;  10,  the 
straight  collecting  tubule,  lined  with  columnar  cells; 
n,  the  excretory  duct,  lined  with  tall  columnar  cells. 


HISTOLOGY.  113 

The  sinus  of  the  kidney  is  formed  by  the  union  of  the 
excretory  duct  and  is  lined  with  white  fibrous  tissue  of 
the  tubules,  and  is  continuous  with  the  ureters. 

Hirer cise  No.  61. — Make  sections  of  the  tissue  given 
you  and  study,  mount  and  preserve. 

URETERS. 

The  ureters  are  the  two  tubes  that  convey  the  urine 
from  the  kidneys  to  the  bladder  and  are  composed  of 
three  coats  ;  viz :  mucous,  muscular  and  fibrous.  They 
are  lined  with  squamous  cells. 

THE  BLADDER. 

The  bladder  is  composed  of  the  same  number  of 
coats  as  the  ureters.  It  is  lined  with  squamous  cells. 

THE  URETHRA. 

The  urethra  consists  of  two  coats  lined  in  different 
parts  with  different  cells — in  the  prostatic  portion  with 
transitional  cells;  the  middle  portion  with  stratified 
columnar;  and  in  the  penile  portion  with  columnar 
cells. 


CHAPTER  XV. 


THE  REPRODUCTIVE  ORGANS. 


The  reproductive  organs  of  the  male  are  the  testes, 
prostate  glands,  Cowper's  gland  and  the  penis. 


THE 

The  testes  are  composed  of  tubular  glands,  and  pro- 
duce the  spermatozoa.  They  are  covered  with  a  cap- 
sule of  fibrous  tissue.  The  -coverings  of  the  testes  are 
tunica  vaginalis,  tunica  albuginea  and  tunica  vascu- 
losa.  These  coats  dip  down  into  the  organ  and  divide 
it  into  lobes.  The  lobes  are  composed  of  convoluted 
tubules,  straight  tubes  and  a  net  work  of  tubes.  The 
convoluted  tubules  are  lined  with  four  layers  of  epithe- 
lial cells,  beginning  on  the  basement  membrane,  and 
coming  outwards.  You  see  the  parietal  cells,  then  the 
mother  cells,  then  the  spermatoblasts  and  finally  the 
spermatozoa  —  all  these  layers  are  found  in  the  lumen 
of  the  convoluted  tubule.  And  the  spermatozoa  are  the 

(114) 


HISTOLOGY. 


115 


T.   S.   TESTICLE, 

Showing  capsule,  septa  of  connective  tissue  and  lobules, 
generating  cells.  The  straight  tube  and  the  network  of 
tubes  are  a  continuation  of  the  same  tube.  These  tubes 
all  unite  before  they  leave  the  epididymis  and  form  the 
duct  of  the  testes,  called  vas  deferens.  It  consists  of 
three  coats,  (a  very  thick  muscular  coat).  The  vasa 
efferentia  makes  up  the  epididymis.  The  globus  major 
is  the  head  of  the  epididymis  and  the  globus  minor  is 
the  tail  of  the  same.  The  vas  deferens  constitutes  the 
spermatic  cord.  The  hard  mass  of  fibrous  tissue  upon 
which  rests  the  net  work  of  tubules  is  called  the  medi- 


116  LABORATORY  METHODS  OF 

astinum.  The  spermatozoon  is  a  long,  comma  shaped 
body.  It  is  divided  into  three  parts:  head,  middle 
piece  and  tail.  The  vesicitla  seminal  is  is  the  expanded 
portion  of  the  vas  deferens  in  addition  to  having  a  few 
mucous  glands  of  tubular  type  in  its  mucous  mem- 
brane. "The  blood  supply  enters  the  tunica  vasculosa 
and  mediastinum." — Leroy.  Lymphatics  are  found  in 
two  locations :  in  the  tunica  albuginea  and  in  the  space 
between  the  tubules.  The  nerves  are  of  both  varities 
and  are  found  in  the  spaces  between  the  seminiferous 
tubules. 

THK   PSNIS. 

The  penis  is  composed  of  two  parts,  the  corpora 
cavernosa  and  the  corpus  spongiosum,  having  a  large 
amount  of  connective  tissue  between  them.  The  blood 
supply  is  derived  from  the  dorsal  and  the  corpora  cau- 
ernosa  arteries.  The  lymphatics  empty  into  the  deep 
lymphatics  of  the  pelvis.  The  nerves  are  of  both 
varieties.  They  are  derived  from  the  pudic  and  hypo- 
gastric  plexus  of  the  sympathetic.  The  nerves  end  in 
nerve  corpuscles  or  end  bulbs.  The  glands  of  the  gen- 
erative organs  are  of  the  tubular  type. 

THK  OVARIES. 

The  ovaries  are  the  female  organs  of  generation, 
placed  one  on  each  side  in  the  pelvic  region.  They  are 


HISTOLOGY.  117 

covered  with  two  capsules :  the  germ  epithelial  layer  of 
cells,  derived  from  the  peritoneum  and  a  tough  band 
of  fibrous  tissue,  called  tunica  albuginea.  The  sub- 
stance of  the  ovary  is  divided  into  two  parts,  the  corti- 
cal and  medulla,  which  merge  into  each  other.  In  the 
young  ovary  one  of  the  cells  of  the  germ  epithelial 
layer  becomes  cut  off  and  drops  down  into  the  medulla. 


L.    S.    OVARY.— CAT. 

1,  germinal  epithelium;  2,  immature  Graafian  follicles; 
S,  stroma;  4,  blood  vessels  in  stroma;  5,  a  Graafian  follicle 
from  which  the  ovum  has  escaped;  6,  7,  ova  in  different 
stages  of  development;  8,  a  fully  developed  ovum;  9,  cor- 
pus -luteum. 


118  LABORATORY  METHODS  OP 

A  band  of  fibrous  tissue  from  the  stroma  encloses  this 
cell.  This  band  is  called  theca  follicnli,  and  is  divided 
in  two  parts,  an  inner  and  an  outer.  The  first  cell  is 
called  primordial  ovum.  Just  inside  of  the  theca  fol- 
liculi  is  seen  a  granular  zone,  called  membrana  granu- 
losa.  Inside  of  this  stratum  is  the  discus  proligerus. 
Now,  inside  of  all  this  is  the  ovum,  a  large  circular 
body,  enclosed  in  two  cell  walls  ;  viz  :  the  zona  pellucida, 
and  the  vitclline  membrane.  In  this  space  between  the 
ovum  and  the  vitelline  membrane  is  found  the  vitellus. 
Situated  to  one  side  of  the  litellus  is  located  the  ger- 
minal vesicle.  In  the  center  of  the  germinal  vesical 
is  the  germinal  spot.  A  Graafian  follicle  that  has  lost 
its  ovum  is  called  a  corpus  luteum.  The  blood  vessels 
enter  the  ovary  at  the  attachment  of  the  broad  liga- 
ments and  pass  into  the  theca  folliculi.  The  lymphat- 
ics are  most  numerous .  in  the  medulla.  The  nerves 
are  of  both  varieties,  and  supply  the  follicles. 
THE:  FAi,u)piAN  TUBES. 

The  fallopian  tubes,  as  all  other  tubes,  consist  of 
three  coats.  The  mucous  coat  in  these  tubes  is  lined 
with  ciliated  epithelial  cells.  The  blood  vessels  are 
the  same  as  in  the  other  cases. 

THE  UTERUS. 

The  uterus  is  the  same  in  structure,  only  the  muscu- 
lar tissue  predominates  in  this  organ.  The  Nabothian 


HISTOLOGY.  119 

glands  are  in  the  mucous  coat  of  the  uterus.     They  are 
called  the  uterine  glands. 

THE  VAGINA. 

The  vagina  is  lined  with  stratified,  pavement  epithe- 
lial cells.  The  muscle  tissue  is  arranged  in  two  layers, 
an  inner  (circular)  and  outer  (longitudinal),  and  is 
composed  of  the  smooth  variety.  The  region  of  the 
clitoris  and  the  labia  minora  contains  many  sebaceous 
glands.  In  the  region  of  the  urethra,  mucous  glands 
are  found  called  Bartholin's  glands. 


CHAPTER  XVL 


THE  RESPIRATORY  SYSTEM. 


This  system  comprises  the  nose,  epiglottis,  trachea, 
larynx,  bronchi,  lungs  and  pleura.  These  tissues  are 
derived  from  the  mesoderm  and  the  epiderm.  The 
nerve  supply  is  from  the  cerebro  spinal  and  sympathetic 
system.  The  epiglottis  is  chiefly  yellow  cartilage.  Its 
mucous  surface  is  covered  sparingly  with  taste  buds. 
The  larynx  is  cartilagenous.  The  trachea  is  composed 
of  three  coats :  mucous,  submucous  and  fibrous.  The 
mucous  coat  consists  of  ciliated  epithelial  cells,  base- 
ment membrane  and  elastic  fibers.  The  submucous 
coat  consists  of  cartilage,  glands,  elastic  tissue  and  non- 
striated  muscle.  The  cartilage  is  of  the  hyaline  vari- 
ety and  is  arranged  in  the  shape  of  a  horseshoe.  "The 
large  bronchi  are  like  the  trachea  in  structure,  only  the 
cartilage  is  not  arranged  in  a  loop,  but  is  arranged  in 
plates." — Sobotta.  A  bronchus  having  a  diameter  not 
over  I  micron  is  called  a  bronchiole.  They  still  have  a 
layer  of  muscle  and  a  layer  of  cubic  epithelial  cells. 
These  bronchioles  lead  into  the  respiratory  bronchioles, 
(120) 


HISTOLOGY. 


121 


LUNGS. 

and  these  in  turn  lead  into  the  alveolar  ducts.  The 
alveolus  is  divided  into  separate  chambers,  all 
communicating  with  one  opening,  called  an  in- 
fundibular space.  The  alveolus  is  lined  with  a 
single  layer  of  epithelial  cells.  The  blood  vessels  fol- 
low the  course  of  the  bronchus  and  break  up  into  cap- 
illaries around  the  alveolus,  and  there  the  blood  under- 
goes the  gaseous  change.  The  blood  changes  its 
name  from  venous  to  arterial.  The  lymphatics  and 
nerves  are  scattered  all  in  the  substance  of  the  lung 
tissue.  The  nerves  are  of  both  varieties,  and  they  fol 
low  the  course  of  the  blood  vessels. 


122  HISTOLOGY. 

Exercise  No.  62. — Make  T.  S.  of  the  lungs. 

Note  the  technic. 

Observe  all  the  points  in  the  tissue. 


CHAPTER  XVII. 


THE  SPECIAL  SENSES. 


The  special  senses  are  located  in  the  nose,  eye, 
tongue,  skin  and  ear. 

THE  NOSE. 

The  mucous  membrane  of  the  nose  is  divided  into 
two  tracts,  the  respiratory  and  the  olfactory.  The  ol- 
factory is  the  tract  that  furnishes  the  cell  in  which  the 
nerves  end  that  carry  the  sensation  of  odor  to  the  cen- 
ter of  olfaction  or  smell.  It  consists  of  two  layers  of 
epithelial  cells.  The  cells  that  receive  the  sensation 
are  very  long  and  nucleated.  The  cells  in  which  the 
sensation  is  generated  are  placed  beneath  a  layer  of 
tall,  columnar  cells.  The  nerves  are  of  both  kinds. 
The  special  nerves  communicate  with  the  olfactory 
center.  The  sensory  nerves  do  not  come  in  contact 
with  the  olfactory  cells.  They  are  derived  from  the 
trifacial,  while  the  others  are  derived  from  the  ol- 
factory nerve.  The  blood  vessels  are  found  in  the 
sub-epithelial  layer  ending  in  capillaries.  The  lym- 
phatics are  numerous. 

(123) 


124 


LABORATORY   METHODS  OF 


THE  EYE. 

The  eye  is  the  organ  of  vision  and  consists  of  a  hol- 
low globe  divided  into  two  chambers :  the  anterior  and 
posterior,  separated  by  the  iris  and  lens.  In  front  of 
the  anterior  chamber  is  the  cornea  and  behind  is  the 
lens.  The  posterior  chamber  is  composed  of  the 
sclera,  choroid  and  retina.  The  sclera  is  the  white  of 
the  eye.  The  choroid  is  the  coat  between  the  retina 
and  the  sclera.  The  retina  is  the  vision-producing 
membrane.  The  cornea  is  composed  of  five  layers. 


SECTION  OF  RETINA. 
1,  pigment  layer;   2,  layer  of  rods  and  cones. 


HISTOLOGY.  125 

The  lens  is  a  biconvex  body,  placed  between  the  an- 
terior and  posterior  chambers  of  the  eye,  and  held  in 
place  by  the  suspensory  ligaments.  The  anterior  cham- 
ber is  filled  with  a  clear  fluid  called  aqueous  humor, 
and  the  posterior,  with  a  fluid  called  vitrous  humor. 
The  lymphatics  are  found  around  the  nerves  of  the 
eye  and  in  a  few  other  places.  The  nerves  are  derived 
from  the  ciliary  nerves.  The  retina  contains  ten  layers. 

1.  The  layer  called  internal  limiting  membrane. 

2.  The  layer  of  nerve  fibers. 

3.  The  layer  of  ganglion  cells. 

4.  The  inner  molecular  layer. 

5.  The  nuclear  layer. 

6.  The  outer  molecular  layer. 

7.  The  outer  nuclear  layer. 

8.  The  external  limiting  membrane. 

9.  The  layer  of  rods  and  cones. 
10.  The  layer  of  pigment  cells. 

Make  sections  of  tissue  supplied,  according  to  direc- 
tions. 

THE  EAR. 

The    special    organ    of    the  sense  of  hearing  is  lo- 
cated in  the  ear.    The  ear  is  of  rnesodermic  origin. 


126 


LABORATORY  METHODS  OF 


TONGUE:. 


The  tongue  is  the  special  organ  of  the  sense  of  taste, 
and  is  placed  in  the  oral  cavity.  It  is  covered  with  a 
layer  of  stratified  epithelial  cells.  The  mucous  mem- 
brane studded  with  the  little  projections  called,  papilke, 
upon  which  you  find  little  taste  buds.  The  m.uscle 
layers  run  in  every  direction.  It  is  well  supplied 
with  blood  vessels  and  lymphatics.  The  nerves  are  of 


SECTION  OP  THE  TONGUE. 

1,  mucosa,  showing  three  papillas,  covered  with  squamous 
epithelial  cells;  2,  stratified  epithelium;  3,  taste  buds;  4 
muscular  layer;  5,  adipose  tissue. 


HISTOLOGY.  127 

the  sensory  and  taste  variety.  The  papillae  are  of  three 
varieties,  filiform,  fungiform  and  circumvalate.  They 
all  have  a  few  taste  buds  connected  with  them.  The 
tongue  is  divided  into  halves  by  the  septum  linguae. 
In  the  tongue  will  be  found  several  tissues  of  the 
body,  viz :  fat,  muscle,  epithelial  cells  and  fibrous 
tissue. 

Exercise  No.  63. — Make  T.  S.  of  the  tongue  and  ex- 
amine. 


PART  II. 

URINALYSIS 


INTRODUCTION. 


It  always  gives  me  pleasure  to  give  my  experience 
in  our  chosen  profession,  especially  when  it  is  in  any 
way  connected  with  the  analysis  of  urine,  a  subject 
which  I  think  is  of  vital  importance  in  the  diagnosis  of 
diseases,  since  by  it  we  may  arrive  at  a  definite  conclu- 
sion as  to  the  nature  and  location  of  the  pathological 
state.  The  older  physicians  for  many  years  depended 
entirely  on  a  sugar  and  albumin  test  of  urine.  But 
that  no  longer  satisfies  the  investigating  mind  of  the 
scientific  physician,  since  sugar  and  albumin  are  only 
two  of  the  many  pathological  elements  found  in  the 
urine.  The  methods  of  discovering  the  constituents 
are  few  and  simple,  and  most  of  the  methods  require 
only  a  short  time  to  complete  them;  so,  you  see,  for 
time  spent  and  money  expended  on  one  hand  and 
knowledge  gained  and  scientific  treatment  established 
on  the  other,  there  is  no  sort  of  comparison.  Now, 
gentlemen,  the  value  of  these  analyses  will  depend  on 
our  knowledge  of  pathology. 

Pathology  is  only  perverted    Physiology.     Since  we 
know  that  the  urine  is  the  sewage  of  the  body  and  that 

(131; 


132  INTRODUCTION. 

it  is  the  principal  way  by  which  the  soluble  material 
of  the  blood  and  tissues  leave  the  economy,  we  naturally 
look  to  the  urine  to  find  any  pathologic  changes  that 
might  be  taking  place  in  the  body.  These  changes 
are  of  two  varieties :  i .  An  increase  in  the  normal 
constituents  of  the  urine.  2.  An  introduction  into  the 
urine  of  some  foreign  matter  that  may  be  acting  as  a 
poison  ;  in  fact,  anything  introduced  into  urine  that  is 
not  normally  found  in  it  is  a  poison,  illustrated  in  the 
case  of  poisons,  as  treated  in  Toxicology.  This  variety 
is  of  most  interest  to  the  specialist.  The  first  variety 
is  oi  most  interest  to  the  physician,  since  in  ordi- 
nary diseases  it  is  either  an  increase  or  decrease  of  the 
normal  constituents  of  the  urine.  An  analysis  of  the 
urine  not  only  gives  us  an  idea  of  the  changes  going 
on  in  the  urinary  tract,  but  in  other  places.  A  chem- 
ical examination  is  essential,  since  it  gives  an  idea  of 
the  changes  going  on  in  the  body  a  distance  from  the 
urinary  tract,  while  the  microscope  only  detects  the 
solids  in  the  specimen.  How  .  are  these  changes  de- 
termined ?  We  are  now  going  to  talk  about  the  most 
importiant  constituent  of  the  urine,  since  by  it  the  nitro- 
genous material  leaves  the  body. 


URINALYSIS. 


The  analysis  of  the  urine  is  very  important  to  the 
physician  and  surgeon.  It  consists  of  three  depart- 
ments, viz. :  chemical,  physical  and  microscopical.  In 
order  to  gain  any  information  from  an  analysis,  it  is 
necessary  to  understand  the  secretion  of  the  urine. 
There  are  two  theories  as  to  the  secretion  of  it,  viz. : 
the  capsule  of  Bowman,  acting  as  a  filter,  separates 
the  urine  from  the  blood ;  the  epithelial  cells  lining  the 
uriniferous  tubules  secrete  a  part  of  the  urine.  In 
fact,  both  processes  go  on  at  the  same  time.  The  first 
is  Ludwig's  theory,  and  the  second  is  Bowman's.  The 
experiment  of  Heidenhain  settled  the  question  of  the 
epithelial  cells  having  a  part  in  the  secretion  of  the 
urine.  He  injected  a  coloring  matter  into  the  veins  of 
an  animal.  After  a  suitable  time  he  made  sections  of 
the  kidneys  of  the  animal,  and  found  the  coloring  mat- 
ter in  the  cells  of  the  uriniferous  tubules,  but  none  in 
the  capsule  of  Bowman.  Other  experiments  prove  the 
theory  good,  for  when  a  patient  has  uremia,  the  urinif- 
erous tubules  have  lost  their  epithelial  lining,  as  shown 
by  the  microscope  on  analyzing  the  urine.  The  theory 

of  Lurwig  is  proven  by  the  change  in  the  amount  of 

(133) 


134  LABORATORY  METHODS  OF 

urine  after  drinking  a  large  amount  of  water.  The 
urine  is  increased  in  quantity  but  not  in  quality.  (For 
the  histology  of  the  kidney,  see  the  illustration  of  that 
organ.)  The  urine  is  the  sewage  of  the  system, 
and,  therefore,  contains  the  results  of  all  the  tissue  me- 
tabolism. The  changes  the  tissues  undergo  in  the 
body  you  understand  from  the  knowledge  of  physiol- 
ogy. The  urine  being  a  filtrate  of  the  blood,  it  would 
be  natural  to  suppose  that  it  was  of  the  same  reaction 
as  the  blood ;  but  not  so.  It  is  acid,  while  the  blood  is 
alkaline.  The  cause  of  this  change  was  proven  by 
C.  H.  Ralf  to  be  the  result  of  vital  phenomena.  The 
solutions  necessary  for  an  analysis  of  the  urine  are 
hydrochloric  acid,  sulphuric  acid,  acetic  acid,  nitric 
acid,  liquor  potassii,  liquor  sodii,  liquor  sodium  aqua 
carbonate,  liquor  baric  chloride,  ammoniae  and  liquor 
magnesium  sulphate,  Hain's  solution,  silver  nitrate 
solution,  liquor  plumbic  acetate,  alcohol,  water  and 
hypobromite  solution. 

The  apporatus.  A  notebook,  two  dozen  test-tubes 
and  a  test-tube  rack,  two  conical  glasses,  spirit  lamp, 
beakers  and  watch  glasses,  funnels,  2,000  c.c.  vessel, 
graduates,  filter  paper,  water  bath,  tripod,  swab  for 
cleaning  the  test  tubes,  a  microscope  and  several  pi- 
pettes. Other  things  will  be  added  as  we  go  on  with 
the  work. 


URINALYSIS.  135 

The  following  solution  should  be  made  by  the  phy- 
sician: hypobromite  solution  and  Haines'  solution. 

The  others  can  be  purchased  at  a  drug  store.  The 
first  is  prepared  as  follows :  25  c.c.  of  water  to  100 
grams  of  sodium  hydrate ;  the  bromine  and  sodium  hy- 
drate must  be  kept  "in  separate  vessels.  When  ready 
to  make  the  test,  take  10  c.c.  of  the  soda  solution  and 
i  c.c.  of  the  bromine,  add  equal  amount  of  water,  mix, 
then  put  the  whole  into  the  ureometer,  then  add  the 
urine. 
Technic. 

1.  Put  the  solution  in  the  ureometer,  filling  the  arm 
and  one-half  of  the  bulb. 

2.  Add  i  c.c.  of  urine  to  the  solution ;  use  a  pipette. 

3.  Read  the  amount   of   urea   in   the   specimen  by 
means  of  the  graduate  on  the  arm  of  the  instrument. 
This  reading  represents  the  amount  in  24  hours  in  a 
liter  of  urine.     The  normal  amount  ranges  from  20  to 
33  grams.     The  urea  is  decreased  or  increased  in  many 
pathological  conditions  of  the  system.     (For  details 
on  the  subject,  see  Practice,  and  for  source  of  urea,  see 
Physiology;  for  composition  and  for  reaction  between 
the  solution  and  the  urea,  see  Medical  Chemistry.) 

In  urinalysis  all  vessels  must  be  clean.  For  chem- 
ical analysis,  the  specimen  must  be  that  of  the  urine 
representing  the  24  hours  urine,  if  not  the  whole. 


136  LABORATORY  METHODS  OF 

For  microscopic  examination  it  is  best  to  have  the 
urine  as  fresh  as  possible.  In  the  summer  time  it  is 
best  to  add  to  each  four  ounces  of  urine  10  grains  of 
sodium  salicylate  to  prevent  decomposition. 

The  physical  properties  of  the  urine  are  too  well 
known  to  require  special  mention  here.  (See  Physi- 
ology oh  the  following  points:  color,  odor,  transpar- 
ency, reaction  and  amount. )  The  color  is  straw ;  the 
reaction  is  acid;  the  odor  is  peculiar  to  itself;  the 
amount  ranges  from  1000  c.c.  to  1500  c.c. ;  normal 
urine  is  transparent.  In  disease  all  of  this  is  changed, 
each  change  representing  a  pathological  process. 
Sometimes  several  properties  are  changed  in  one  case. 
(For  diseases  represented  by  the  changes,  see  Prac- 
tice.) The  method  of  making  the  physical  analysis  is 
simple.  Use  the  senses  given  you.  For  reaction 
use  litmus  paper.  Specific  gravity  of  the  urine  is  de- 
termined by  using  the  urinometer.  The  normal  grav- 
ity is  1 020  to  1025. 
Method : 

1.  Fill  the  cylinder  of  the  urinometer  two-thirds. 

2.  Set  it  on  a  level  table,  and  introduce  the  bulb  of 
the  instrument  in  the  center  of  the  cylinder;  when  the 
bulb  comes  to  rest,  read  off  the  graduate  on  the  stem  of 
the  bulb.     In  case  you  have  not  enough  urine  to  find 
the  gravity,  you  must  add  water  enough  to  float  the 
bulb ;  then  multiply  the  gravity  of  the  mixture  by  the 


URINALYSIS.  137 

sum  of  the  water  and  urine  together.  This  is  not  prac- 
ticable only  in  case  the  urine  is  of  high  gravity. 

The  specific  gravity  of  the  urine  depends  upon  the 
solids  in  it.  The  most  abundant  solid  is  the  urea;  the 
other  solids  are  in  the  shape  of  salts— therefore,  the 
acids  and  metals  are  in  the  combined  state.  The  tests 
ior  the  metals  are  the  same  as  the  ones  used  in  analytical 
chemistry;  this  is  also  true  with  the  acids.  The  de- 
composition of  urine  is  accompanied  with  a  peculiar 
odor,  caused  by  the  urea  breaking  up  into  its  constit- 
uents. The  elimination  of  the  urea  represents  the  tis-- 
sue  metabolism,  a  process  which  is  explained  under  the 
head  of  Metabolism  in  this  text.  The  urea  represents 
about  two  per  cent,  of  the  solids,  and  the  solids  repre- 
sent about  four  per  cent,  of  the  urine.  "Schroder's  ex- 
periment is  as  follows :  he  injected  into  the  liver  by  the 
portal  vein  a  mixture  of  ammonium  carbonate  and 
blood,  and  on  examining  the  blood  in  the  hepatic  vein, 
it  was  found  to  contain  urea  in  abundance.  This  does 
not  occur  when  the  same  experiment  is  performed  with 
any  other  organ  of  the  body,  so  that  his  experiment 
proves  the  great  importance  of  the  liver  in  urea  for- 
mation."— Kirk. 

The  accumulation  of  urea  in  the  body  is  called  urae- 
mia, and  the  same  may  be  said  of  the  accumulation  of 
uric  acid ;  only  the  term  is  uricacidaemia.  There  is 


138  LABORATORY  METHODS  OF 

some  discussion  as  to  the  relation  between  urea  and 
uric  acid.  The  ratio  between  them  is  as  one  to  forty- 
five.  (For  further  information  see  Medical  Chemistry.) 
At  certain  times  of  the  day  the  reaction  of  the  urine 
differs  widely.  The  time  when  it  is  acid  is  called  the 
acid  tide,  and  when  it  is  alkaline  it  is  called  the  alkcb- 
line  tide. 

What  are  the  most  abundant  constituents  of  the 
urine  ? 

What  is  said  of  the  acids  and  metals  in  the  urine? 

What  is  the  cause  of  the  odor  of  stale  urine? 

Examine  the  spread  of  urine  after  the  most  im- 
proved method. 

How  do  you  make  a  microchemic-analysis  ? 

UREA. 

With  the  ureometer  make  a  test  for  urea. 

What  is  the  normal  per  cent,  of  urea? 

What  does  urea  represent  in  the  body  ? 

Give  Schroder's  theory  in  reference  to  the  formation 
of  urea,  as  taught  by  an  experiment. 

Where  is  urea  formed  in  the  body  ? 

What  is  urea  accumulation  in  the  blood  called? 

Is  there  any  connection  between  urea  and  uric  acid? 

What  is  an  accumulation  of  uric  acid  in  the  blood 
called? 


URINALYSIS.  1 39 

How  is  uric  acid  formed  for  experimental  purposes? 

Take  10  c.c.  of  urine  and  2  c.c.  of  hydrochloric  acid ; 
put  the  acid  in  the  urine  and  set  aside  for  two  hours. 
Examine  under  the  microscope. 

Note  the  shape  of  the  crystals. 

What  is  meant  by  the  acid  tide  and  the  alkaline  tide? 

MICROCHEMIC  ANALYSIS. 

Take  a  drop  of  urine  to  be  examined  and  put  it  on 
the  slide ;  also  a  drop  of  the  reagent  to  be  used.  Place 
it  on  the  slide  near  the  urine,  put  a  cover-glass  on  the 
drops,  and  watch  the  reaction. 

Set  a  method  by  which  you  will  always  work  in  your 
own  office  or  laboratory. 

ABNORMAL,  CONSTITUENTS  IN  THE  URINE. 

Albumin  in  the  urine  is  the  first  to  consider.  Albu- 
min is  coagulated  by  heat  and  certain  chemicals,  so  we 
make  use  of  these  principles  in  our  analysis. 

Exercise  No.  i. — Secure  your  key  and  clean  out  your 
locker.  Keep  your  locker  locked.  Note  the  quan- 
tity of  a  liter  as  the  demonstrator  measures  it  out. 

Each  student  should  collect  the  urine  for  24  hours 
from  a  patient  and  bring  it  to  the  laboratory  to  analyze. 


140  LABORATORY  METHODS  OF 

Keep  the  urine  in  a  tightly  stoppered  bottle  to  prevent 
evaporation.  From  the  total  quantity  for  the  whole 
twenty-four  hours  samples  should  be  taken  to  make 
tests. 

SPECIFIC   GRAVITY. 

With  the  urinometer  take  the  gravity  of  the  urine 
and  note  the  same.  Dilute  the  urine  one-half  and  take 
the  gravity  again.  Note  the  result.  Give  the  range  of 
gravity  in  normal  urine.  Name  what  conditions  will 
increase  the  gravity ;  also  what  conditions  will  decrease 
the  same.  (See  Physiology.) 

NORMAL  COI/DR. 

What  is  the  normal  color  of  urine?  Give  the  cause 
of  the  same.  Note  the  color  of  ten  specimens  of  urine. 
(Read  Physiology  on  the  urine.) 

REACTION    OF    URINE. 

The  reaction  of  urine  is  due  to  what?  Under  what 
circumstances  is  it  alkaline?  How  do  you  test  the 
reaction  of  the  urine? 

COMPOSITION  OF  URINE. 

Total  grams 1,500.000 

Water 1,440.000 


URINALYSIS.  141 

Solids 60.000 

Urea 35-°°o 

Uric    acid -75° 

Sodium  chloride 16.500 

Phosphoric  acid  3-5°° 

Sulphuric  acid   2.000 

Ammonia    .625 

Creatinin .900 

Chlorine I  i.ooo 

Potassium   ' 2.500 

Sodium 5-5°o 

Calcium .260 

Magnesium   .210 

TEST  FOR  CHLORIDES  IN  URINE. 

Take  10  c.c.  of  urine,  add  to  it  I  drop  of  potassium 
chr ornate,;  add  to  this  silver  nitrate  until  the  color 
changes  to  red,  noting  the  amount  of  silver  added.  Now 
calculate  the  amount  of  chlorine  present.  I  c.c.  of  sil- 
ver nitrate  corresponds  to  .00586  grams  of  sodium  chlo- 
ride. A  more  practical  method  is  to  take  a  small 
amount  of  urine  and  add  to  it  I  drop  of  I  in  8  silver 
nitrate  solution.  Note  results.  If  a  curdy  precipitate 
occurs,  which  is  not  divided  on  shaking  the  urine,  the 


142  LABORATORY  METHODS  OF 

quantity  is  normal,  or  if  it  is  divided  easily,  the  quan- 
tity is  diminished. 

The  presence  of  albumin  in  small  quantity  does  not 
make  a  change  in  the  reaction  of  the  chemicals.  (For 
clinical  significance,  see  Tyson  on  Urine.) 

PHOSPHATES  IN   URINE. 

To  10  c.c.  of  urine  add  a  small  amount  of  ammonia. 
Warm  gently  until  the  earthy  phosphates  begin  to 
separate.  Set  aside  10  or  15  minutes  until  they  are 
completely  settled ;  then  measure  the  height  of  the  pre- 
cipitate, and  if  it  is  i  mm.  high,  the  quantity  is  normal. 
(For  clinical  see  Tyson  on  Urine.)  This  is  the  test 
for  the  earthy  phosphates. 

THE  AUCAUNE  PHOSPHATES 

are  determined  by  adding  to  the  urine  a  small  quantity 
of  magnesium  fluid,  which  precipitates  the  phosphates 
into  a  cloudy  precipitate.  This  cloud  is  normal.  The 
more  the  cloud,  the  more  the  phosphates. 

SULPHATES. 

To>  a  small  amount  of  urine  add  a  drop  of  barium- 
chloride,  and  if  a  cloudiness  is  produced  it  indicates 
that  a  normal  quantity  of  sulphates  are  present.  If  a 


TTRINALYSIS.  1 43 

curdy  precipitate  is  produced,  it  indicates  an  increased 
quantity.     (For  clinical,  see  Tyson  on  Urine.) 

1.  Make  a  standard  solution  of  sodium  phosphate 
(10.885  grains  of  well  crystallized  sodium  phosphate) 
in  distilled  water  and  dilute  to  a  liter.     (50  c.c.  then 
contains  .01  centigram  of  phosphoric  acid. 

2.  Make  a  saturated  solution  of  potassium-ferrocy- 
anide. 

3.  Make  a  solution  of  sodium  acetate  by  dissolving 
10  grams  of  sodium  acetate  in  100  c.c  .of  acetic  acid 
c.  p.  and  diluting  the  1,00  c.c.  with  water. 

4.  Make  a  solution  of  uranium  acetate,  such  that  I 
c.c.  will  correspond  to  .005  milligrams  of  phosphoric 
acid  made  as  follows :  Dissolve  the  uranium  acetate  in 
water  until  the  proper  strength  is  obtained. 

Step  i.  Take  50  c.c.  of  standard  solution  of  sodium 
phosphate  in  a  beaker  with  5  c.c  of  the  solution  of  so- 
dium acetate.  Heat  to  90  degrees  C. 

Step  2.  Let  the  uranium  solution  run  in  until  the 
warm  mixture  ceases  to  precipitate.  This  is  done  from 
a  burette. 

Step  3.  Take  a  small  amount  of  the  potassium  so- 
lution and  place  it  on  a  clean  white  dish ;  then  transfer 
a  drop  of  the  warm  mixture  to  the  dish  containing  the 
potassium  solution.  If  the  reddish  brown  precipitate 


144  LABORATORY  METHODS  OF 

does  not  appear,  continue  the  adding  of  the  uranium 
solution  until  it  does.  Then  read  off  the  amount  used. 
This  is  the  amount  that  will  precipitate  o.i  decigram 
of  phosphoric  acid.  Now  make  a  quantity  of  the 
uranium  solution,  say  a  liter. 

SULPHATES. 

Make  a  solution  so  that  i  c.c.  of  the  solution  of  bari- 
um chloride  will  precipitate  12.50  milligrams  of  sul- 
phuric acid  prepared  as  follows : 

1.  Solution..   Dissolve  30.50  grams  of  barium  chlo- 
ride in  a  liter  of  water. 

2.  Solution  the  same  strength  as  the  one  above,  but 
a  different  solution.     Dissolve  27.57  grams  of  potas- 
sium sulphate  in  a  liter  of  water. 

CHLORIDES. 

1.  Make  a   saturated   solution   of  potassium   chro- 
mate. 

2.  Make  a  solution    of    silver    nitrate    such    as   I 
c.c.   will   precipitate    10   milligrams   of   NaCl,   as   fol- 
lows :     Dissolve  29  grams  of  stick  silver  nitrate  in  a 
liter  of  water. 

ABNORMAL    URINE. 

Sugar  solution.  30  grains  of  copper  sulphate  dis- 
solved in  one-half  ounce  of  water;  then  add  one-half 


URINALYSIS.  145 

ounce  of  glycerine.  Thoroughly  mix.  To  this  mix- 
ture add  5  ounces  of  liquor  potassse.  Make  i  liter  of 
this. 

ALBUMIN. 

Albumin  is  one  of  the  abnormal  constituents  of  the 
urine.  The  presence  of  the  same  is  indicative  of 
some  lesion  in  the  kidneys,  especially  in  the  uriniferous 
tubules,  according  to  Bowman's  theory.  This  may  be 
a  nephritis.  The  method  of  finding  the  presence  of 
the  same  is  simple.  It  depends  upon  the  fact  that 
albumin  is  coagulated  under  certain  conditions.  These 
we  must  now  study.  Heat  the  urine  at  the  top  for  a 
short  while  and,  if  albumin  is  present,  there  will  be  a 
white  ring  at  the  junction  of  the  cold  and  hot  urine, 
which  is  the  test  for  albumin. 

Make  an  acid  test  for  albumin.  Take  a  small  quan- 
tity of  nitric  acid,  and  place  it  in  a  test-tube  and  al- 
low a  small  amount  of  urine  to  flow  down  the  side  of 
the  tube.  If  albumin  is  present,  there  will  be  a 
white  ring  or  zone  at  the  point  of  contact.  This  is 
said  to  detect  i  part  in  100,000  parts  of  urine.  But 
this  is  not  as  delicate  as  the  heat  test. 

GRAVIMETRIC   METHODS. 

Gravimetric  methods  are  of  extreme  importance  to 
the  physician  in  cases  of  Bright' s  Disease.     But  the 
10 


146  LABORATORY  METHODS  OF 

method  is  so  long  that  a  busy  physician  has  not  time 
to  follow  it ;  so  we  must  depend  on  some  approximate 
methods,  the  best  of  which  are  to  precipitate  the  albu- 
min by  means  of  boiling  the  urine  and  adding  a  few 
drops  of  nitric  acid  and  setting  it  aside  for  twelve 
hours.  Shake  the  specimen  once  or  twice  in  order  to 
get  a  uniform  mixture  of  the  materials  in  the  urine. 
A  more  accurate  way  is  to  take  an  Esbach's  albumi- 
nometer.  (See  your  own  drowings  for  the  same.) 

Method :  Take  the  specimen  of  urine  and  put  it 
into  the  apparatus  to  the  graduation  U,  then  put  in  the 
reagent  to  the  letter  R ;  put  in  the  stopper ;  shake  well ; 
set  aside  until  next  period;  read  off  the  amount  of 
albumin  on  the  albuminometer. 

Each  graduation  denotes  i  gfam  of  dried  albumin 
in  a  liter  of  the  urine. 

The  next  method  is  to  use  the  centrifuge,  which  is 
very  simple  to  use,  and  is  very  accurate  in  results. 
Learn  to  use  the  centrifuge. 

The  amount  of  albumin  found  in  a  specimen  is  very 
variable,  but  not  over  two  per  cent,  in  any  case.  This 
is  a  very  large  amount. 

Sugar  in  the  urine  is  the  next  important  abnormal 
ingredient.  The  detection  of  the  same  depends  on  the 
power  that  sugar  has  of  reducing  the  copper  salts  into 
cuprous  hydrate,  which  is  yellow  or  reddish  yellow. 


URINALYSIS.  147 

Hain's  Solution  is  the  best  to  use  in  the  test.  The  mere 
change  is  not  conclusive  of  the  presence  of  sugar, 
since  a  reddish  brown  is  the  test  for  sugar — that  is,  the 
color  desired,  but  we  may  judge  from  the  color  to  a 
certain  extent,  whether  sugar  is  present  or  not. 

FERMENTATION  TEST. 

The  fermentation  test  is  performed  as  follows: 
Take  a  saccharometer  and  place  in  it  a  small 
part  of  a  cake  of  yeast  and  fill  it  up  to  the  bulb  with 
the  urine  to  be  tested;  set  aside  until  the  next  period, 
and  read  off  the  amount  of  sugar  in  twelve  hours  in 
a  liter  of  the  urine.  The  graduations  on  one  side  of 
the  apparatus  represent  grams  to  the  liter;  on  the 
other,  grains  to  the  ounce. 

PRACTICAL    QUESTIONS    IN    NORMAL    URINALYSIS. 

Give  the  histology  of  a  urine-bearing  tube. 

Give  the  theory  of  Ludwig  in  regard  to  the  secretion 
of  the  urine. 

Give  C.  H.  Ralf's  experiment  on  the  cause  of  the 
change  in  reaction  between  the  urine  and  the  blood. 

Give  the  histology  of  a  glomerulus  of  the  kidney. 

Give  the  theory  of  Bowman  in  regards  to  the  secre- 
tion of  the  urine. 

Give  Heidenhain's  theory  of  secretion  of  urine  as 
shown  by  an  experiment. 


148  LABORATORY  METHODS  OF 

Give  the  conclusions  of  the  same  experimenter. 
How  did  you  determine  the  amount  of  water  in  the 
urine  ? 

How  much  urine  voided  in  twenty- four  hours? 
How  much  urea  in  a  sample  of  normal  urine  for 
twenty- four  hours? 

How  did  you  make  the  urea  test?     Describe  in  de- 
tail the  process. 

What  solution  did  you  use? 

What  are  the  most    abundant    constituents    of    the 
urine? 

Give  the  formula  for  urea. 

Give  the    test  for    chlorides  in    urine,    phosphates, 
lution. 

Give  the  properties  of  urea. 

Give  the   test   for   chlorides   in   urine,   phosphates, 
urates,  sulphates,  sugar,  albumin  and  bile. 

What  is  the  color,  odor,  reaction  and  specific  grav- 
ity of  normal  urine? 

1  Name  and  describe  the  apparatus  used  in  determin- 
ing the  above. 

Give  cause  of  each  property. 

What  is  the  consistence  of  normal  urine? 

Give  five  causes  why  the  urine  may  be  cloudy. 


URINALYSIS.  149 

Give  the  test  for  nucleo-albumin. 

Give  the  source  of  peptones  in  the  urine;  also  test. 

Give  the  test  for  bile  in  the  urine. 

Where  is  bile  made  in  the  body? 

Give  the  indican  test. 

What  is  the  presence  of  indican  in  the  urine  called? 

MICROSCOPIC   URINALYSIS. 

Take  a  spread  of  the  urine  that  you  used  when  you 
precipitated  the  earthy  phosphates  and  place  it  on  the 
slide  and  examine. 

Method:  With  a  long  pipette,  take  some  of  the 
urine  from  the  bottom  of  the  sedimentation  glass  and 
place  it  on  the  slide,  cover  with  a  cover-glass,  using 
just  enough  urine  to  fill  the  space  between  the  cover 
glass  and  the  slide.  Focus  and  make  drawings  of 
what  you  see.  (See  the  Atlas.)  Wipe  off  the  slide, 
cover,  and  receive  the  spread  that  the  assistant  will 
give  you ;  examine.  (See  Atlas.)  Take  a  small  quan- 
tity of  urine  and  set  aside  for  a  while  to  settle  and  use 
the  pipette  on  the  same. 

What  method  of  making  a  spread  is  this? 

Take  the  same  amount  of  urine  £nd  do  the  same  with 
it.  Only  use  the  centrifugal  apparatus. 

Why  did  you  use  the  centrifuge? 

Find  the  reaction  of  the  urine  you  used  in  finding 
the  earthy  phosphates. 


LABORATORY  METHODS  OF 

Find  the  specific  gravity  of  the  urine;  note  the  dif- 
ference between  the  normal  gravity  and  this  one. 

What  is  the  cause  of  the  decrease  in  gravity? 

Dilute  the  same  urine  to  one-third  and  find  gravity 
again. 

Find  the  reaction  of  the  urine  you  brought  the  first 
day,  also  the  gravity. 

What  has  taken  place  in  the  sample  since  you  have 
been  studying  it-? 

In  case  you  have  not  enough  urine  to  find  the  grav- 
ity, how  will  you  proceed  to  find  it? 

What  is  the  gravity? 

Test  the  sample  of  urine  for  urea  after  the  hypo- 
bromite  method. 

What  is  the  percentage  of  urea? 

What  ureometer  do  you  use,  and  what  advantage 
has  it  over  other  kinds? 

What  is  the  formula  for  urea? 

Give  the  reaction  between  the  solution  and  the  urea. 

EXAMINATION    OF    SEDIMENT. 

Make  a  test  of  the  sediment  in  the  urine  that  has 
become  cloudy  from  standing  twenty-four  hours,  and 
determine  the  nature  of  the  sentiment. 


URINALYSIS.  151 

Method :  Pour  in  the  tube  some  of  the  urine  and  heat 
it,  and  if  a  cloudy  precipitate  appears,  the  sediment  is 
due  to  earthy  phosphates.  If  the  cloud  remains  after 
the  addition  of  an  acid,  it  may  be  due  to  albumin ;  if  it 
disappears,  it  may  be  due  to  the  presence  of  phos- 
phates; if  it  disappears  on  the  addition  of  heat  alone 
it  is  due  to  urates;  if  on  the  addition  of  an  acid  the 
piecipitate  disappears  with  the  evolution  of  gas,  the 
precipitate  is  due  to  carbonates. 

What  is  the  cause  of  ammoniacal  odor  in  urine  that 
has  been  standing  for  a  while  at  a  moderate  tempera- 
ture? 

What  causes  the  white  precipitate  that  forms  at  the 
bottom  of  the  vessel? 

How  can  you  tell  when  the  alkalinity  is  due  to  a 
fixed  alkali  or  to  a  volatile  alkali? 

Method :  Test  a  sample  of  urine  with  litmus  paper, 
then  dry  the  paper,  and  if  the  blue  remains,  the 
alkalinity  is  due  to  a  fixed  alkali;  if  the  blue  disap- 
pears, it  is  due  to  a  volatile  alkali. 

NUCI<£0- ALBUM  IN. 

Test  for  nucleo-albumin.  This  is  a  very  simple  test. 
It  is  performed  as  follows :  Take  a  small  quantity  of 
the  urine  to  be  tested  and  dilute  the  sample  to  reduce 
the  salts  that  hold  the  albumin  or  mucin,  which  can 


152  LABORATORY  METHODS  OP 

be  differentiated  by  simply  boiling  it  with  a  mineral 
acid.  If  a  substance  is  produced  that  is  reduced  by 
cupric  oxid  the  substance  is  mucin;  if  not  reduced,  it 
is  nucleo^albumin. 

PEPTONES. 

Test  for  peptones  in  the  urine:  Place  a  drachm 
of  Fehling's  Solution  in  a  test-tube,  and  put  a  small 
amount  of  urine  in  the  tube,  and  if  peptones  are  pres- 
ent a  halo  of  a  rose-colored  hue  will  float,  forming  the 
test  for  peptones.  .(For  the  pathology  for  peptonura 
see  Practice  of  Medicine.) 

SUGAR  TEST. 

Moore's  Test  for  sugar  in  the  uf ine :  Take  a  small 
quantity  of  urine  in  a  test-tube  and  one-half  as  much 
strong  alkali  and  boil,  and  if  a  reddish-brown  or  yel- 
lowish-brown precipitate  appears,  sugar  is  present. 
This  precipitate  intensifies  as  the  boiling  is  continued. 

HYDROBILIRUBIN. 

Test  for  hydrobilirubin  or  urobilin :  Add  a  small 
quantity  of  ammonia  to  the  urine  to  be  tested  and  fil- 
ter the  same.  To  the  filtrate  add  a  little  chloride  of 
zinc,  and  if  a  green  color  appears,  urobilin  is  present. 


URINALYSIS.  153 

UROERYTHRIN. 

To  a  small  amount  of  urine  to  be  examined,  add  a 
small  amount  of  neutral  acetate  of  lead,  and  if  a  yel- 
low color  appears,  uroerythrin  is  present.  (See  Ty- 
son for  clinical  significance  or  the  Practice  of  Medi- 
cine.) 

URATES. 

What  is  the  brick-dust  deposit  in  the  urine? 

The  murexide  test  for  urates  is  a  practical  test. 
Place  a  little  urates  in  a  tube  and  evaporate  the  same 
after  adding  a  little  nitric  acid.  A  yellow  residue  is 
left.  Add  a  little  ammonia  and  the  residue  turns  vio- 
let, which  forms  the  test  for  the  urates  or  uric  acid. 
On  the  addition  of  potassium  solution  the  color  be- 
comes blue. 

Give  the  formula  for  ammonium  carbonate,  also 
ammonium  carbamate.  (See  Medical  Chemistry.) 


Test  for  bile  in  the  urine  :  Take  a  small  amount  of 
urine  and  put  it  in  a  test-tube.  Now  let  a  small 
amount  of  commercial  nitric  acid  run  down  the  side 
of  the  tube  on  the  urine.  If  bile  coloring  matter  is 
present,  a  play  of  colors  will  be  seen  at  the  point  of 


154  LABORATORY  METHODS  OF 

contact  of  the  urine  and  the  acid  (green,  blue,  violet, 
red  and  yellow).  This  is  Gmelin's  Test  for  Bile.— 
Tyson. 

BLOOD. 

This  is  a  common  occurrence.  The  presence  of  the 
same  is  detected  by  the  color  and  the  use  of  the  mi- 
croscope. The  cause  of  the  blood  in  the  urine  is  a 
common  question  asked  the  physician.  It  is  caused 
as  follows:  i.  Local  or  renal  congestion.  2.  Trau- 
matism  of  the  ureters,  bladder,  calculi,  acute  cystitis, 
and  urethritis,  simple  and  specific.  3.  General  dis- 
eases, such  as  malarial-fever.  The  color  of  the  urine 
is  the  chief  index  to  the  beginning  of  the  analysis. 
The  microscope  comes  in  next.  It  is  the  surest  method 
to  find  the  corpuscles  in  the  urine  before  you  say  you 
are  dealing  with  a  case  of  Haematuria.  Since  there 
are  so  many  other  elements  that  resemble  blood  in  color 
it  is  not  safe  to  go  by  the  color  alone.  There  are 
many  tests  for  blood,  chemically,  but  these  are  not  ab- 
solute, so  we  will  not  spend  the  time  on  them.  (See 
Practice.) 

PYURIA. 

The  presence  of  pus  in  the  urine  is  called  pyuria. 
There  are  two  tests  for  pus  in  the  urine.  1st.  Any 


URINALYSIS.  155 

strong  alkali  added  to  the  urine  will  give  a  ropy  pre- 
cipitate. 2.  Any  good  microscope  will  give  you  a  fair 
outline  of  pus  corpuscles,  which  is  the  best  test  for 
a  busy  physician. 

What  causes  pus  in  the  urine?  Any  lesion  of  the 
urinary  tract,  where  pus  is  being  formed,  will  result 
in  a  deposition  of  pus  in  the  urinary  tract.  (These 
conditions  are  many.) 


PART  HI. 

BACTERIOLOGY 


BACTERIOLOGY. 


Bacteriology  is  the  science  of  the  minute  organisms 
that  inhabit  the  sea,  land  and  atmosphere — in  fact, 
every  place  that  can  be  inhabited.  Their  morphology 
is  simple.  They  are  of  three  general  classes,  viz.: 
rods,  spheres  and  curves. 

Classify  the  spheres  into  cocci :  Monococci,  *.  e., 
one  sphere;  diplococci,  two  spheres;  streptococci, 
spheres  arranged  in  a  chain;  staphylococci,  spheres  ar- 
ranged in  a  bunch.  Staphylococci  are  divided  into 
several  names,  according  to  the  color  of  the  growth 
on  the  medium. 

GENERAL    BIOLOGY   OF   BACTERIA. 

There  are  six  points  to  consider  in  the  cultivation 
of  bacteria:  food  supply,  reaction,  moisture,  gaseous 
surroundings,  temperature  and  light. 

We  will  first  consider  the  food  supply,  which  -is  the 
medium  on  which  they  grow.  It  is,  in  nature,  the 
different  substances  that  they  accidently  fall  on  and 
grow ;  but,  in  the  laboratory,  it  is  the  different  media 

that  we  make  for  their  sustenance  or  growth.     Bac- 

(159) 


160  LABORATORY  METHODS  OF 

teria  serve  a  useful  purpose  in  nature  by  breaking  up 
the  complex  substances  that  we  find  in  nature  into 
simpler  ones. 

The  reaction  of  the  medium  is  of  importance,  since 
many  bacteria  will  not  grow  on  any  but  an  acid  me- 
dium, and  all  but  a  very  few  require  an  alkalin  me- 
dium. 

Moisture  is  the  next  to  consider.  All  bacteria  re- 
quire moisture;  but  some  can  live  with  less  than  oth- 
ers; some  can  stand  drying  for  several  days  and  then 
remain  vital  when  placed  in  a  suitable  place  for 
growth  (for  an  example,  take  the  anthrax). 

The  relation  of  bacteria  to  the  gases  of  the  atmos- 
phere is  another  important  consideration.  Some  can 
live  without  the  oxygen,  while  others  cannot  live  even 
a  short  while  without  it;  still  another  variety  can  live 
with  or  without  the  presence  of  air. 

Temperature  is  the  next  to  be  considered.  The 
point  where  the  bacteria  will  grow  best  is  called  the 
optimum  temperature.  This  is  usually  the  same  as 
that  of  summer  weather.  The  temperature  in  which 
they  grow  with  the  greatest  difficulty  is  called  the 
minimum  temperature. 

MEDIA. 

1.  Take  beef  extract 3  grams 

2.  Peptone 10  grams 


BACTERIOLOGY.  161 

3.  Sodium  chloride , , .  > 5  grams 

4.  Water   1000  c.c. 

Boil    until    ingredients    are    dissolved     (fifteen     to 

twenty  minutes)  ;  add  water  from  time  to  time  to 
keep  up  the  1 ,000  c.c.;  allow  to  cool  slightly;  meas- 
ure out  1,000  c.c.,  and  place  them  in  a  large  flask. 

Determine  the  Reaction. — Process:  Take  phenolph- 
thalein  as  an  indicator  and  normal  20  sodium  hydrox- 
ide as  an  alkali ;  take  5  c.c.  of  the  medium  and  40 
c.c.  of  distilled  water;  put  12  drops  of  the  indicator 
into  the  mixture  and  proceed  to  add  the  alkali  from  the 
burette  until  the  indicator  shows  sufficient  quantity  of 
the  alkali,  which  can  be  observed  by  watching  the  color 
of  the  fluid  as  it  changes  from  a  colorless  to  a  red. 
Calculate  from  this  how  much  sodium  hydroxide  will 
be  required  to  alkalinize  the  whole  liter  of  medium; 
then  add  and  boil  forty-five  minutes.  Keep  up  the 
level  by  adding  water;  filter  and  distribute  in  the  ves- 
sels you  are  going  to  use.  Sterilize  fifteen  minutes  each 
day  for  three  successive  days  in  the  Arnold  sterilizer. 

PEPTONE  SOLUTION. 

Take  sodium  chloride,  0.5  grams;  take  peptone,  i.oo 
gram;  water,  100  c.c. 

We  will  make  other  media  from  time  to  time  as  they 

are  needed.     Everything  is  sterilized. 
31 


162  LABORATORY  METHODS  OF 

Agar  and  gelatine  are  simply  bouillon  hardened,  or 
stiffened  by  them. 

Everything  must  be  labelled  in  the  laboratory  ac- 
cording to  the  model  shown  you. 

Examine  an  inoculating  needle,  draw  and  learn  the 
use  of  the  same  in  more  than  one  method. 

Notice  the  demonstrator,  as  he  inoculates  one  tube. 
Describe  in  full  the  process  in  your  notebook. 

Inoculate  several  agar  and  gelatin  tubes,  some  on  a 
slant  surface  and  some  on  a  square  surface. 

It  is  necessary  that  the  glassware  be  thoroughly 
cleaned  before  it  is  used.  Several  methods  have  been 
suggested,  but  the  best  is  the  one  which  follows: 
First,  take  cleaning  fluid  and  wash  the  vessels  in  if. 
The  fluid  is  made  as  follows :  dissolve  80  grams  of 
potassium  bichromate  in  300  c.c.  of  warm  water; 
when  cooled,  slowly  mix  the  solution  by  constant  stir- 
ring with  450  grams  of  sulphuric  acid ;  store  the  liquid 
in  a  glass  bottle  with  glass  stopper. 

CLEANING   TEST-TUBES. 

Wash  them  in  warm  soapsuds;  then  place  them  in 
the  cleaning  fluid, twenty  minutes;  then  pour  the  solu- 
tion back  into  the  bottle ;  wipe  the  tubes  dry  and  drain 


BACTERIOLOGY.  163 

in  the  sink  on  the  drain-board;  wash  them  until  the 
color  of  the  solution  disappears. 

FLASKS   AND    PETRI   DISHES. 

Wash  them  with  soap  and  water ;  then  fill  them  with 
the  fluid  and  allow  it  to  remain  ten  minutes.  Proceed 
as  in  the  other  case. 

COVER   GLASSES   AND   SLIDES. 

Drop  the  cover  glasses  in  the  fluid  one  by  one  and 
let  remain  ten  minutes.  Take  them  out  and  wash 
them  in  alcohol;  then  place  them  in  the  sterilizer  at  a 
temperature  of  160  degrees  C  for  one  hour.  If  a  drop 
of  bouillon  is  spread  on  a  cover  glass,  the  drop  will 
spread  out  evenly  if  the  cover-glass  is  clean. 

PLUGGING  TUBES. 

-This  is  a  simple  process,  but  much  depends  on  the 
way  you  do  it.  The  plug  should  be  neatly  made  and 
firmly  rolled  just  large  enough  to  fit  the  test-tube  or 
flask.  They  are  usually  made  of  cotton-batting,  which 
should  be  sterilized  before  being  used.  The  plug 
must  not  be  too  large  or  too  small.  It  is  best  to  have 
them  just  large  enough  to  fit  the  tube  or  flask  and  no 
larger.  The  whole  must  be  sterilized. 


164  LABORATORY  METHODS  OF 

METHOD   OF   STERILIZING. 

Place  the  vessels  in  the  sterilizer  and  light  the  gas; 
watch  the  thermometer;  keep  the  temperature  up  to 
135  degrees  C  for  one  hour;  then  shut  off  the  gas  and 
let  the  temperature  go  down  to  45  degrees  C,  and  then 
open  the  door  and  take  the  vessels  out.  Store  in  the 
locker  till  wanted. 

Light  is  another  factor  in  the  growth  of  bacteria. 
It  is  a  very  decided  germicide  to  certain  bacteria. 
It  is  probably  the  ultra  violet  and  green  rays  that  do 
the  killing  of  the  germs.  But  light  and  heat  both  are 
more  certain  to  kill  them  than  either  heat  or  light 
alone. — Muir  and  Ritchie. 

Movement  of  Bacteria. — This  is  variable.  Some 
will  move  wlien  they  are  in  the  act  of  reproducing. 
In  what  is  commonly  called  the  spore  stage  some 
move  more  rapidly  than  at  other  times ;  some  less. 
Some  substances  will  cause  bacteria  to  move  towards 
them,  such  as  salts  of  potassium  and  sodium.  This 
property  is  called  CHemiotaxsis,  positive  and  negative. 

The  Part  Played  by  Bacteria  in  Nature. — As  has 
been  said,  the  function  of  bacteria  is  to  break  up  the 
complex  molecule  into  more  simpler  ones,  as  shown 
by  the  souring  of  milk,  the  curdling  of  cheese  and 
the  fermentation  of  sugar  solution. 


BACTERIOLOGY.  165 

Saprophytic  and  Parasitic  Bacteria. — The  bacteria 
that  grow  on  dead  organic  matter  is  called  sapro- 
phytic,  the  variety  that  grow  on  living  organic  matter 
is  called  parasitic. 

The  variability  of  bacteria  is  of  much  importance, 
since  the  line  of  demarcation  is  not  yet  definitely  de- 
termined. 

The  death  of  bacteria  is  usually  determined  by  the 
fact  that  in  a  medium  in  which  they  were  growing 
luxuriantly,  on  inoculating  a  new  supply  of  the  same 
medium  they  fail  to  grow. 

Those  agents  which  kill  the  greatest  number  of  bac- 
teria in  the  shortest  time  are  carbolic  acid  and  bi- 
chloride of  mercury  (the  first  in  a  solution  of  I  to 
1,000;  the  second  in  a  solution  of  I  to  20.) 

These  are  called  germicides.  Those  agents  that  so 
alter  the  medium  that  bacteria  will  not  grow  on  it  are 
called  antiseptics. 

The  best  germicides  are  heat,  acids  and  the  heavy 
metals  in  solution.— Muir  and  Ritchie. 

CULTIVATION    OF  BACTERIA. 

In  order  to  study  bacteria,  it  is  necessary  to  have 
them  growing  apart  from  other  bacteria.  In  nature 
this  is  not  so  since  they  are  mixed  up  in  general  in  the 
medium;  it  is  only  in  the  blood  and  tissues  that  we 


166  LABORATORY  METHODS   OF 

find  a  few  in  the  isolated  state.  When  we  have  suc- 
ceeded in  obtaining  a  pure  culture,  we  have  only  taken 
the  germ  from  its  mixed  state  and  have  it  growing  on 
a  medium  that  is  suited  to  its  growth  and  development. 
These  pure  cultures  are  absolutely  necessary  to  a  suc- 
cessful study  of  the  germ  in  question. 

The  Study  of  the  Methods  of  Sterilization.— Heat 
is  the  best,  whether  in  the  form  of  dry  or  moist  heat. 
This  is  the  manner  in  which  we  do  it :  take  a  platinum 
wire  and  heat  it  to  a  bright  red,  then  let  it  cool  to  a 
red  heat;  it  is  now  ready  to  be  inserted  into  the  cul- 
ture tube. 

All  instruments  are  sterilized  in  the  bunsen  before 
they  are  used.  The  glassware  is  sterilized  by  a  dry 
heat.  This  is  done  by  the  use  of  the  hot-air  oven. 
Watch  the  demonstrator  use  the  oven. 

The  method  of  using  the  oven  is  simple  and  will 
be  used  a  great  deal  before  we  are  through  this  work. 
This  method  is  not  used  in  sterilizing  the  media  as  it 
will  evaporate  the  fluid  media  and  scorch  the  solid  be- 
fore it  will  reach  the  temperature  135  degrees  C. 

Sterilizing  by  Moist  Heat. — Boil  the  apparatus  for 
five  minutes  in  a  covered  vessel,  and  that  will  insure 
sterilizing  for  almost  any  variety  of  bacteria,  but  will 
not  kill  spores;  so  we  boil  the  material  about  twenty 
minutes  for  three  successive  days.  Steam  sterilis- 


BACTERIOLOGY.  167 

ers  are  used  in  the  following  manner :  in  using  the  Ar- 
nold and  the  Koch  Sterilisers,  put  the  media  in  the 
steriliser  at  the  same  time  you  put  in  the  water,  and 
that  will  insure  the  media  getting  the  same  amount  of 
heat  that  the  water  gets.  Boil  the  whole  fifteen  min- 
utes for  three  successive  days.  This  is  Tyndall's  in- 
termittent method.  Each  of  these  will  be  demonstrated 
in  the  class. 

Sterilisation  by  steam  at  high  pressure  is  a  quick 
method  but  is  not  adapted  to  media  work.  It  is  car- 
ried on  by  means  of  the  autoclave.  Sterilisation  by 
low  pressure  is  adapted  to  one  kind  of  media — that  is, 
blood-serum,  on  Tyndall's  principle. 

Plating  of  Bacteria. — Take  a  sterilised  platinum 
wire ;  let  it  cool  slightly ;  then  introduce  it  into  the  col- 
ony of  bacteria  and  then  into  tube  No.  I ;  shake 
well;  then  sterilise  again;  take  a  loop  full  from 
tube  No.  i  and  inoculate  tube  No.  2  with  the  same; 
sterilise;  take  a  loop  from  tube  No.  2  and  inoculate 
tube  No.  3 ;  pour  the  contents  into  the  same  number  of 
Petri  dishes  and  number  them  according  as  the  tubes 
were  numbered;  set  aside  until  next  day  and  note  the 
change  if  any.  This  will  be  explained  as  we  go  along 
in  our  work.  Always  sterilise  your  needle  before  and 
after  use.  (The  media  must  be  melted.) 

Hanging-Drop  Culture. — This  is  made  by  simply 
taking  the  drop  of  media  containing  the  growth  and 


168  LABORATORY  METHODS  OF 

placing  it  on  the  top  side  of  a  cover-slip  and  turning 
the  cover-slip  down  'on  the  slide  and  watching  the  mo- 
tion or  non-motion  of  the  germ. 

COIyONY    COUNTING. 

1.  Take  a  Petri  dish  and  divide  it  off  into  equal  seg- 
ments and  place  it  on  the  table,  with  a  black  surface 
under  the  dish. 

2.  Take  I  c.c.  of  the  fluid  to  be  used  and  carefully 
mix  it  with  a  tube  of  gelatine. 

3.  Pour  the  contents  of  the  tube  into  the  dish. 

4.  Incubate  twenty-four  hours  at  the  room  temper- 
ature. 

5.  With  the  aid  of  a  lens  count  the  colonies  that  ap- 
pear in  the  dish.   (Each  colony  represents  a  bacterium 
to  begin  with.) 

BACTERIOLOGICAL   EXAMINATION   OF   THE   BLOOD. 

1.  Sterilise  the  part  carefully  with  1-1,000  perchlo- 
ride  of  mercury ;  then  wash  off  the  mercury  with  alcu- 
hol;  let  the  alcohol  evaporate;  wipe  the  part  with  a 
dry  sterile  cloth  or  absorbent  cotton. 

2.  Prick  the  part  with  a  needle  prepared  for  the 
purpose  by  sterilising  with  dull  heat. 

3.  Take  a  loop  of  the  blood  from  the  part  and  put 
it  in  the  media,  and  if  you  get  a  growth,  make  the 
whole  test  for  bacteria  as  directed  further  on. 


BACTERIOLOGY.  169 

Urine  and  water  are  examined  in  the  same  way  (in 
general  the  vessels  are  cleaned). 

Filtration  of  bacteria  is  an  important  process  in  the 
study  of  the  organisms.  It  is  done  by  means  of  a  por- 
celain tube  which  is  the  only  perfect  filter  known. 
Watch  the  demonstrator  use  the  filter. 

Examine  water  for  Bacteria. — Take  the  water  and 
place  it  in  the  plating  dish  and  after  a  time  count  the 
colonies. 

Method:  Take  a  definite  quantity  of  water  and 
put  it  in  a  dish  of  agar-agar;  put  the  same  in  an 
incubator  for  twenty- four  hours;  take  out  and  count 
the  colonies  with  the  aid  of  a  lens.  Each  colony  re- 
presents a  single  germ. 

Practical  Use  of  the  Analysis. — First,  we  find  the 
B.  Coli  and  B.  Typhosus  in  water.  The  earth  is  an- 
other place  where  we  find  a  great  number  of  bacteria, 
i.  e.,  the  B.  tetani. 

DETERMINING    THE    THERMAL    DEATH     POINT    OF    BAC- 
TERIA. 

This  is  a  simple  procedure.  Take  some  bouillon 
and  inoculate  it  with  some  living  bacteria;  place  it  in 
a  water  bath  and  introduce  a  thermometer;  turn  on 
the  gas  and  note  the  temperature;  when  the  tempera- 
ture reaches  the  body  temperature  take  out  one  of 


170  LABORATORY  METHODS  OF 

the  tubes  and  inoculate  a  new  tube  with  the  contents 
of  the  hot  tube ;  wait  a  while  and  take  out  another  tube 
and  proceed  as  in  the  above  case,  and  so  on,  until  you 
have  taken  out  several  tubes,  all  at  a  different  tem- 
perature. Set  the  whole  aside  until  the  next  period 
and  note  which  one  has  the  growth  on  it.  It  will  usual- 
ly be  the  one  that  stays  in  the  bath  the  shortest  time 
which  will  give  the  growth.  The  ones  on  which  there 
is  no  growth  are  dead  and  the  temperature  at  which 
they  died  is  known  by  the  reading  on  your  tube,  since 
you  noted  the  temperature  when  you  took  out  each 
eye  of  bouillon  to  inoculate  the  new  tube. 

DETERMINE   THE   STRENGTH   OF   ANTISEPTICS   AND   GER- 
MICIDES. 

Take  some  of  the  culture  and  subject  it  to  the  action 
of  different  strengths  of  the  germicides  in  a  Petri  dish 
Any  germ  will  serve  for  the  test,  or  if  the  agent  is  a 
gas,  we  will  place  some  pieces  of  blotting  paper,  which 
have  been  soaked  in  some  good  medium,  and  scatter 
them  in  a  closed  chamber  and  set  the  gas  generator 
going.  After  a  while  take  some  of  the  paper  and 
smear  the  surface  of  a  sterile  medium  and  the  growth 
will  not  take  place  if  the  germicide  is  strong  enough. 

INOCULATING  ANIMALS. 

The  mouse,  guinea  pig,  rabbit,  and  white  rat  are  the 
ones  that  are  used  most.  Take  the  animal  and  sterilise 


BACTERIOLOGY.  171 

the  parts  to  be  inoculated;  then  take  an  eye  of  the 
growth  on  a  needle  after  having  made  the  incision  at 
the  root  of  the  tail  of  the  animal ;  inoculate  the  animal 
and  keep  him  in  a  cage  to  prevent  him  from  spreading 
the  disease,  if  it  is  pathogenic.  There  are  other  meth- 
ods. Fill  a  sterile  hypodermic  and  inoculate  the  animal 
in  the  dorsal  veins  of  the  ear  and  observe  the  same  pre- 
cautions as  in  other  cases.  This  determines  the 
pathogen etic  or  non-pathogenetic  properties  of  the  bac- 
teria. 

Staining  bacteria  is  a  very  important  step  in  the 
study  of  bacteria.  We  will  take  that  up  when  we 
come  to  stain  the  several  kinds  of  bacteria. 

Diagnosing  bacteria  is  very  difficult  and  is  not  free 
from  error.  But  we  do  it  this  way:  we  observe  the 
color  or  growth  of  the  medium  on  which  they  grow 
best.  Their  action  in  the  hanging-drop  preparation, 
their  morphology,  their  mode  of  bunching,  their 
source,  their  relation  to  air,  their  chemical  production, 
their  action  on  an  animal  (pathogenetic  property), their 
reaction  with  Gram's  stain  and  analine  dyes. 
These  are  the  points  by  which  we  make  a  diagnosis  of 
a  colony  of  bacteria.  A  few  others  we  will  discuss  as 
we  come  to  them. 

COVSR-GLASS  PREPARATIONS. 

These  are  simple  to  make,  but  we  must  be  careful 
not  to  contaminate  the  growth  when  we  take  the  eye 


172  LABORATORY  METHODS  OP 

from  the  tube  to  make  the  spread  on  the  cover-glass. 
This  is  prevented  by  taking  the  needle  and  heating 
it  to  a  bright  red  in  the  flame  of  the  bunsen,  and  let- 
ting it  cool  a  little  before  placing  it  in  the  culture  or 
growth.  The  spreading  of  the  material  on  the  cover 
and  drying  in  the  air,  and  then  passing  it  three  times 
times  through  the  flame,  then  applying  the  stain,  ac- 
cording to  the  special  process  that  we  will  learn  as  we 
go  along,  constitute  the  technic. 

METHOD  OF  EXAMINING  THE  MATERIAL  THAT  A  PHY- 
SICIAN  MAY  BRING  TO  YOU  TO  EXAMINE. 

Several  cover  preparations  must  be  made;  one  to 
be  stained  with  Gram's  method,  another  to  be  stained 
with  carbolfuchsin,  one  with  gentian  violet,  and  an- 
other with  a  mordant  to  stain  the  spores,  if  any  are 
present. 

Gelatin  plates  should  be  made  and  kept  at  room 
temperature.  Now,  if  no  growth  has  appeared  on  the 
plates  in  twenty-four  hours,  it  is  well  to  try  some  oth- 
er medium.  But,  if  a  growth  does  occur,  you  must 
take  a  tube  for  your  research  that  has  only  about  200 
colonies  on  it.  Now  the  first  question  to  decide  is, 
Are  all  the  colonies  of  one  or  of  many  species?  The 
question  is  answered  by  noting  the  characteristics  of 
the  different  species.  This  is  not  always  known.  Note 


BACTERIOLOGY.  173 

all  yoii  can  from  the  plate,  and  all  you  can  from  the 
hahging-drorj  culture,  and  all  you  can  from  the  mi- 
crtfecdpe  arid  the  reaction  oh  certain  media,  also  re- 
action on  certain  animals.  The  technic  will  be  dis- 
cussed in  this  chapter. 

MICROSCOPIC    EXAMINATION    OF    AN    UNKNOWN. 

Make  the  spread  of  the  twenty-four  hour  culture; 
note  the  form,  the  size  and  the  appearance  of  the  pro- 
toplasmic contents  regarding  its  reaction  to  a  stain ;  the 
method  of.  grouping. 

Has  it  a  capsule? 

Do  the  bacteria  stain  with  a  watery  stain? 

Do  they  require  a  mordant  to  stain  them  or  not? 

How  do  they  behave  toward  Gram's  stain? 

Are  they  motile  ? 

Do  they  form  spores? 

Do  they  possess  flagella? 

What  is  the  best  temperature  for  their  growth? 

Growth  on  Media :— What  are  the  characteristics  of 
the  growth?  What  is  its  relation  to  oxygen?  What  is 
the  best  temperature  for  its  growth?  Observe  its 
growth  on  gelatine  stab-culture,  rate  of  growth,  form 


174  LABORATORY  METHODS  OP 

of  growth,  both  on  and  in  the  substance,  presence  or 
absence  of  liquefaction,  color,  presence  or  absence  of 
gas  formation,  odor  and  relation  to  reaction  of  media, 
streak  culture,  shake  culture,  plate  culture  and  the 
condition  of  the  colonies.  Note  the  reaction  on  agar 
in  the  same  way,  also  bouillon  and  special  media. 
Note  reaction  on  animals  when  inoculated. 

Inoculating  Animals. — The  animal  is  either  a  rat, 
mouse,  guinea-pig,  or  rabbit.  Method :  Take  the  ani- 
mal, and  sterilize  the  part  selected  for  the  operation, 
and  inoculate  the  same  and  watch  results.  If  the  ani- 
mal dies  in  twenty-eight  days,  the  germ  is  pathogenic. 
But  it  usually  dies  in  less  time.  The  best  location  for 
the  inoculation  is  in  the  dorsal  veins  of  the  ear  or  the 
root  of  the  tail. 

Autopsies  on  Dead  Animals. — Method :  Take  a 
strong  cord,  a  trough  and  the  dead  animal ;  tie  the  ani- 
mal in  the  trough,  soak  the  animal  in  carbolic  acid 
solution  to  prevent  the  dry  hairs  from  flying  in  the 
room  during  the  autopsy,  hence  contaminating  the  air 
of  the  room;  now  open  the  body  and  examine.  The 
animal  should  be  burned  after  the  operation. 

INOCULATION   AND   INCUBATION. 

To  inoculate  a  tube  of  medium,  take  a  culture  and 
place  it  between  the  thumb  and  index-finger;  take  a 
tube  of  the  medium  that  you  are  going  to  inoculate 


BACTERIOLOGY.  175 

and  place  it  between  the  index  and  second  finger ;  take 
an  inoculating  needle  and  sterilize  it  in  the  flame;  let 
it  cool  a  little;  then  place  it  in  the  culture;  after  hav- 
ing taken  out  the  plugs  as  directed  in  the  class  room, 
insert  the  needle  in  the  culture  tube,  then  transfer 
an  eye  to  the  tube  of  medium  .and  replug  both  tubes. 
Pass  the  culture  on  to  the  next.  After  having  ster- 
ilized the  needle,  pass  it  also.  Incubate  the  medium 
tube  for  twenty-four  hours  at  room  temperature  after 
having' labeled  the  tube  as  follows:  name,  date  and 
medium.  , 

Hanging-drop  Preparation. — Take  the  hanging- 
drop  slide  and  make  a  preparation  in  the  usual  way 
and  examine  the  same.  Note  the  following:  shape, 
size,  motility  and  spore-formation. 

Dry  Preparation. — Make  a  dry  preparation  after  the 
usual  manner,  viz :  Take  the  cover  slip  and  clean  the 
same  by  the  use  of  alcohol ;  pass  it  three  times  through 
the  flame ;  make  a  thin  spread  from  the  culture ;  dry 
in  the  air;  pass  over  the  flame  until  it  is  fixed,  then 
place  the  whole  under  the  misroscope  and  examine. 

Watery-Stain  Preparation. — Make  a  spread  in  the 
usual  way  and  then  apply  the  stain  a  few  seconds ;  and 
wash  in  a  glass  of  water;  place  under  the  microscope 
and  examine. 


176  LABORATORY  METHODS  OF 

Spore  Stain. — Make  a  spread  in  the  usual  way  and 
stain  for  spores. 

Flagella  Stain. — Make  the  spread  in  the  usual  way 
and  stain  for  flagella. 

Gram's  Stain. — Place  the  spread  on  the  cover  in 
the  usual  way ;  apply  the  stain  and  note  results. 

This  is  what  the  reagents  give  us  in  regards  to  the 
diagnosis  of  bacteria.  It  is  in  order  to  take  up 
the  appearance  of  cultures  of  a  studied  germ.  Inocu- 
late all  the  media  that  you  have  and  note  the  results 
on  the  same.  Inoculate  them  in  all  the  known  ways; 
i.  e.,  stick,  stab,  smear  and  slant  inoculations. 

Note  all  the  changes  that  will  occur  in  twenty-four 
hours.  This  will  help  you  very  much  in  the  diagnosis 
of  the  germ  in  hand. 

Distribute  the  Media: — Take  three  tubes  of  agar- 
agar,  and  three  tubes  of  gelatine  and  three  of  bouillon. 
Inoculate  one  bouillon  and  two  gelatine,  one  slant  and 
one  stick  in -the  same  way  as  the  gelatine.  Incubate 
these  for  twenty-four  hours.  Note  all  the  reactions 
that  occur  in  that  time. 


QUIZ. 


What  are  bacteria? 
Give  their  morphology. 


BACTERIOLOGY.  177 

Where  are  they  found? 

What  are  the  media  on  which  they  grow  best? 

How  do  we  get  rid  of  them? 

What  is  the  best  means  of  getting  rid  of  spores  ? 

How  hot  must  the  medium  be  to  kill  all  known 
spores  ? 

What  is  the  temperature  to  be  obtained  before  you 
can  take  out  the  glassware  when  you  are  sterilizing 
it? 

How  do  you  use  the  hot-air  sterilizer? 

How  do  you  lower  the  temperature  in  the  hot-air 
sterilizer  ? 

Are  media  sterilized  in  the  hot-air  sterilizer? 

What  are  spores? 

What  are  chromatic  granules  ? 

What  do  you  learn  from  the  use  of  the  hanging- 
drop  culture? 

How  did  you  make  the  cell  for  the  culture  ? 
Did  you  find  any  bacilli,  any  cocci,  any  spirilla? 
How  did  you  use  the  moist-sterilizer? 
Why  do  you  use  the  discontinuous  method? 

What  is  meant  by  sterilizing? 
12 


178  LABORATORY  METHODS  OP 

What  is  meant  by  pathogenic,  saprophytic,  chromo- 
genic  and  pyogenic  bacteria? 

Are  bacteria  easily  seen  with  the  ordinary  instru- 
ment? 

At  what  temperature  do  they  grow  best? 
Are  all  bacteria  disease  producers? 
Are  all  bacteria  chromogenic? 
How  did  you  fold  the  filter  for  nitration? 
What  is  the  advantage  in  this  over  the  old  way? 
What  medium  did  you  make? 
Are  the  principles  the  same? 

Take  a  large  tube  of  water  and  sterilize  over  the 
flame. 

How  can  you  tell  when  the  tube  is  sterilized? 
What  is  the  temperature  of  boiling  water  ? 
Are  the  spores  destroyed  in  the  water — if  not,  why 
not? 

If  we  use  a  steam  boiler  can  we  destroy  the  spores? 
If  so,  why  can  we? 

What  is  the  name  of  the  steam  boiler  that  we  use 
in  sterilizing  cultures? 

Name  all  the  agents  used  in  sterilizing,  as  a  class. 
Name  two  methods  of  using  the  dry  heat. 


BACTERIOLOGY.  179 

What  is  the  name  of  the  rod  and  wire  that  you  use 
in  inoculating  the  medium  in  your  hanging-drop 
culture  ? 

How  hot  do  you  make  the  wire? 

What  kind  of  heat  did  you  use  in  sterilizing  the 
media? 

What  kind  of  heat  do  you  use  in  sterilizing  instru- 
ments ? 

Inoculation  of  Tubes. — Watch  the  demonstrator  in- 
oculate a  tube  of  bouillon,  then  do  the  same.  Incu- 
bate it  for  twenty-four  hours. 

Make  a  stained  spread  of  the  material  given  you  and 
examine  the  same. 

Determine  the  class  to  which  the  germs  belong, 
whether  cocci  or  bacilli. 

Consult  the  atlas  for  the  answer  to  the  query. 

Review  the  lesson  you  had  the  last  period ;  note  the 
likeness  or  unlikeness  to  this  lesson. 

Are  all  bacteria  pathogenic  or  not? 

Note  their  color  in  a  growth? 

Make  a  stained  preparation  of  the  germs  at  your 
table  and  examine. 

Note  in  your  notebook  the  points  observed. 

Determine  the  thermal  death-point  of  streptococci 
in  bouillon. 


180  LABORATORY  METHODS  OP 

Method:  Put  some  water  in  the  water-bath;  put 
the  tube  of  culture  in  it,  and  heat  to  56  degrees  C. ;  let 
it  remain  for  fifteen  minutes. 

Inoculate  a  second  tube  from  this  one,  and  store  in 
your  locker.  Note  the  tube  next  day  and  see  if  there  is 
any  growth  in  the  tube.  If  not,  the  thermal  death-point 
is  56  degrees  C.  This  method  is  the  one  we  follow 
at  all  times. 

Learn  the  theory  of  reproduction  of  bacteria.  Note 
the  color  on  the  board  which  represents  the  stained 
germ  and  the  stained  spore. 

tfOOD  SUPPLY. 

The  medium  on  which  bacteria  grows  is  termed 
food.  We  are  to  determine  the  best  food  on  which 
they  grow.  The  best  ones  are  bouillon,  agar-agar  and 
gelatin.  In  order  to  determine  the  reactions  of  the 
medium  after  the  germs  have  been  growing  on  the 
medium,  it  must  be  of  a  composition  the  same  as  that 
of  the  tissues  and  juices  of  the  body,  or  the  same  as 
that  of  the  dead  organic  bodies.  This  we  attempt  to 
do  when  we  make  the  different  media.  The  properties 
of  media  which  must  be  observed  are  clearness  'and  so- 
lidity. The  characteristic  growth  of  the  culture  is  ob- 
served on  solid  media. 


BACTERIOLOGY.  181 

Name  the  processes  you  went  through  when  you 
made  the  stained  preparation  of  the  germs. 

What  is  the  best  method  to  be  followed  when  you 
make  a  preparation  of  bacteria  —  the  stain  or  the  other 
methods  ? 

What  are  the  three  best  stains  for  ordinary  use? 

You  may  use  the  oil-immersion  lens  when  you  make 
a  good  preparation. 

Give  the  preparation  of  some  of  the  stains  in  daily 
use.  .  , 

Make  a  drawing  to  illustrate  the  morphology  of 
bacteria. 

Describe  in  detail  the  method  of  making  a  dry 
spread. 

Watch  the  demonstrator  make  a  plate  of  the  air 
germs. 

Note  the  date  and  medium. 

Note  the  temperature  of  the  room  when  the  plate  is 
made. 

Give  Tyndall's  principle  of  intermittent  sterilizing. 

TYPHOID 


Typhoid  fever  is  an  infectious  disease  caused  by  the 
presence  of  the  B.  Typhosus.    The  B.  was  discovered 


182  LABORATORY  METHODS  OP 

by  Ebert  and  Koch,  in  1880,  and  was  first  secured  in 
pure  culture  from  the  spleen  and  affected  lymphatic 
glands  by  Gaffky,  in  1884. — McFarland. 

The  organism  is  a  short,  small  bacterium,   I  to  20 
.microns  in  length  and  .5  to  .8  microns  in  thickness. 

The  ends  are  rounded  and  sometimes  they  unite  in 
chains,  especially  in  the  potato  growth. 

The  organisms  are  actively  motile,  the  motility  be- 
ing due  to  the  presence  of  a  long  flagellum.  They 
stain  very  well  by  Loffler's  method,  and  they  are  used 
to  demonstrate  the  presence  of  flagella  in  the  study  of 
bacteria  in  the  laboratory.  The  organisms  stain  well 
with  the  ordinary  stains.  They  lose  their  stain  with 
Gram's  stain. 

The  bacillus  has  no  spore;  the  dark  spot  seen  at 
the  end  of  the  germ  is  a  chromatic  granule. 

The  bacterium  is  both  a  parasite  and  a  saprophyte. 
The  bacterium  is  sometimes  present  in  green  vegeta- 
bles which  have  been  sprinkled  with  water  contami- 
nated with  the  organisms.  This  brings  to  mind  a  few 
years  ago  when  the  city  ordered  that  certain  springs  be 
closed  on  account  of  the  prevalence  of  the  bacillus  ty- 
phosus,  and  at  the  same  time  prevented  the  marketmen 
from  sprinkling  their  vegetables  with  water  from  the 
brooks  on  their  way  to  town.  Their  reason  for  such 
action  is  evident  when  we  know  that  the  organisms 


BACTERIOLOGY.  183 

thrive  well  in  water.  Their  resistance  to  all  germicides 
has  been  spoken  of  already.  They  grow  well  at 
room  temperature,  and  boiling  in  water  kills  them, 
since  they  are  killed  at  the  temperature  of  60  degrees  C. 
This  brings  to  mind  another  well-known  fact,  that  the 
people  in  certain  large  cities  are  requested  to  boil  all 
the  drinking  water  before  it  is  used.  We  know  the  rea- 
son why  such  a  course  is  taken  when  we  know  the 
death-point  of  the  germ.  Cold  has  no  effect  on  the 
germ,  and  certain  chemicals  must  be  very  strong  be- 
fore they  will  kill  them.  The  best  chemicals  are  bi- 
chloride of  mercury  and  carbolic  acid.  The  bacilli  are 
killed  in  a  short  time  by  drying.  So  a  hot-air  steril- 
izer will  be  all  right  for  sterilizing  the  vessels  used 
in  the  work  on  this  kind  of  bacteria. 

The  best  method  of  obtaining  the  typhoid  germs  is 
to  take  the  animal  after  it  is  dead  and  get  the  germs 
from  the  spleen  and  lymphatic  gland;  but  you  can  get 
them  from  the  dejecta  of  the  patient  with  difficulty. 

The  bacilli  are  sure  to  be  present  in  the  patient  after 
the  second  or  third  week.  Right  here  is  the  place  to 
try  Widal's  Test. 

The  method  of  making  a  pure-culture  from  a  pa- 
tient's f eces  is  as  follows :  Take  several  tubes  of  gela- 
tin and  melt  the  gelatin ;  have  10  c.c.  of  the  medium  in 
each  tube;  now  add  to  each  tube  i-io  c.c.  of  carboi- 


184  LABORATORY  METHODS  OF 

ic  acid.  In  the  first  tube  put  a  loop  full  of  the  broken 
up  feces  and  take  a  loop  full  from  this  tube  and  trans- 
fer to  tube  number  two  and  so  on.  Next  plate  all  the 
tubes  and  number  them.  The  carbolic  acid  kills  all 
the  saprophytes  and  leaves  only  the  bacillus  typhosus 
and  its  near  congener,  the  bacillus  coli.  The  gelatin 
will  not  be  liquefied. 

For  Widal's  reaction  read  McFarland. 

Serum  diagnosis  has  been  attempted.  This  is  on 
the  principle  of  attenuation  of  germs  by  the  presence 
of  another  species. 

The  Bacterium  Pyocyanus  was  the  antitoxic  growth 
used.  This  is  on  the  principle  of  attenuation  of  germ 
by  the  presence  of  another  species.  This  as  yet  has 
not  come  into  use. 

Make  a  spread  of  bacillus  anthracis. 

What  is  the  morphology  of  the  same? 

Are  they  often  found  in  man? 

Give  the  approximate  diameter  of  the  germ  and  the 
length  in  microns. 

Do  you  observe  the  morphology  of  the  germs  and 
spores  in  them, 

Biology.— The  bacilli  grow  well  on  all  the  ordi- 
nary media— best  grown  at  35  degrees  C. ;  death-point, 
60  degrees  C.  They  withstand  dry  heat  well,  and 


BACTERIOLOGY.  185 

must  be  heated  up  to  a  very  high  degree  before  they 
are  destroyed.  Spores  are  of  the  usual  variety.  The 
action  of  the  gastric  juice  kills  them.  They  effect  ani- 
mals lower  than  man ;  therefore,  they  are  a  disease  for 
the  veterinary  surgeon  to  study.  The  best  method  of 
getting  rid  of  the  dead  animal  is  to  burn  it  or  bury  it 
under  a  pile  of  slacked  lime  several  inches  deep. 

Examine  the  tap  water  for  bacteria  by  making  the 
plate  cultures  in  the  usual  way,  as  follows:  Take  the 
gelatin  and  melt  it  and  pour  it  into  the  Petri  dish; 
then  take  i  c.c.  of  water  from  the  hydrant  and  pour 
it  into  the  dish  on  the  medium,  and  note  results  in  a 
few  days. 

Plate  the  pus  in  the  vessel,  and  examine  the  same 
on  the  slide  after  staining. 

Make  a  stained  preparation  of  the  urine  on  the 
table. 

Tell  to  what  class  the  bacteria  belong,  i.  e.,  if  they 
are  cocci  or  bacilli. 

Some  stain  with  blue  and  others  with  red. 
Make  a  study  of  the  bacillus  of  hog-plague. 
Make  the  usual  cover  preparation;  stain  with  fuch- 
sin;  make  drawings  of  the  same. 

Examine  bacillus  prodigiosus;  note  the  color  of  the 
colonies. 


186  LABORATORY  METHODS  OP 

Use  the  colony  counter  and  make  calculation  on  the 

results. 

Make  a  note  of  the  method  you  would  follow  in 
making  a  bacteriologic  diagnosis,  stating  the  points 
that  you  would  observe  in  each  step. 

Make  a  spread  of  bacillus  tuberculosis  and  stain 
with  fuchsin  and  methyline  blue — the  first,  thirty  min- 
utes ;  the  second,  one-half  minute.  Examine  under  the 
oil  lens. 

Make  a  spread  of  spirillum  cholera  and  stain  with 
fuchsin. 

Tell  how  you  would  determine  the  following  points 
in  the  study  of  a  certain  germ. 
First,  the  pathogenesis  or  non-pathogenesis. 
Second,  the  chromogenesis. 
Third,  the  motility. 
Fourth,  the  morphology. 

Fifth,  the  reaction  to  stains,  and  Gram's  method. 
Sixth,  the  reaction  on  gelatin. 
Seventh,  the  reaction  on  all  media. 
Eighth,  the  source. 

Ninth,  the  germicide  for  the  germs. 
Tenth,  the  thermal  death-point  for  the  germ. 
Eleventh,  the  best  temperature  for  its  growth. 


BACTERIOLOGY.  1 87 

How  do  you  prevent  your  mind  from  letting  slip 

the  points  observed? 

Make  stained  spread  from  the  scrapings  from  your 
mouth;  note  the  morphology. 

Determine  the  death-point  of  streptococci  as  a 
class,  after  the  following  manner :  Put  a  number  oi; 
t.  t.  in  an  incubator  and  heat  the  water  to  56  degrees 
C.  and  let  remain  fifteen  minutes.  Inoculate  a  tube  of 
agar  and  place  it  in  your  locker  until  next  period,  and 
note  if  there  is  a  growth  or  not  on  the  medium.  If 
there  is,  the  germ  or  organism  is  not  killed  at  the  tem- 
perature of  56  degrees  C. 

Study  Streptococcus'  and  Sarcina.  Note  the  effects 
on  bouillon,  and  on  agar-agar,  on  gelatin,  and  on  pota- 
to. (See  Sternberg  on  Streptococcus.) 

See  McFarland  on  biology  of  bacteria  and  note  the 
points  of  interest  to  you  as  a  bacteriologist.  See  what 
he  says  about  the  appearance  of  the  colonies  on  the 
different  media;  note  the  similarity  between  his  de- 
scription and  your  notes.  Note  the  appearance  of  the 
colonies  on  a  gelatin  plate. 

Note  the  color  of  the  growth  of  bacillus  prodigio- 
sus,  sarcina  luteus,  staphylococcus-pyogenes. 

Make  a  plate  of  gelatin  and  inoculate  it  with  a  drop 
of  water  to  see  if  there  are  any  bacteria  in  the  water. 

Study  spore  formation  of  bacteria. 


188  LABORATORY  METHODS  OF 

Study  suppurative  diseases,  their  causes  and  cure. 

Study  the  cause  of  gonorrhoea;  the  method  of  mak- 
ing the  spread  for  the  discovery  of  the  cocci. 

Study  mixed  infection. 

Make  a  cover  spread  of  the  pus  on  the  table  and 
classify  the  germs  in  it. 

INFLUENZA. 

The  disease  Influenza  is  a  widespread  disease  and 
is  more  prevalent  in  winter  than  in  any  other  time. 
It  is  caused  by  the  presence  of  a  certain  bacillus  called 
Bacillus  Influenzae.  They  are  short  rods  1.5  microns  in 
length  and  .3  microns  in  breadth.  The  bacilli  sometimes 
occur  in  chains  and  sometimes  in  bunches.  They  lose 
the  stain  by  Gram's  method.  They  are  non-motile 
and  do  not  form  spores.  They  are  best  stained  with 
carbol  fuchsin.  They  grow  best  on  blood  agar  and 
can  be  grown  best  on  the  agar  by  smearing  the  me- 
dium with  the  sputum  of  the  patient.  The  lower  an- 
imals' blood  is  as  good  as  that  of  the  human  subject. 
The  bacilli  grow  well  at  room  temperature ;  their  opti- 
mum temperature  is  that  of  the  body.  The  power  of 
resistance  of  this  organism  is  of  a  low  order.  The 
mode  of  distribution  of  this  bacillus  is  by  direct  con- 
tact with  the  mucus  and  pus  of  the  person  affected 
with  influenza.  The  location  in  the  body  is  unusally 


BACTERIOLOGY.  189 

in  the  respiratory  organs.  They  are  always  mixed 
with  other  organisms.  The  purest  cultures  can  be  se- 
cured from  the  greenish  yellow-pus  that  may  be  col- 
lected from  the  person  so  affected.  As  the  disease 
advances,  we  may  be  able  to  find  the  germs  in  the 
leucocytes  of  the  blood.  It  is  a  peculiar  fact  that  the 
bacilli  remain  in  the  sputum  for  a  while  after  the  dis- 
ease is  cured. 

Method  of  Making  the  Examination. — Take  some 
of  the  greenish  yellow  pus  and  make  the  usual  cover- 
glass  preparation,  and  stain  the  same  with  carbol- 
fuchsin. 

Give  the  use  of  the  agglutinating  test  of  Widal. 

What  is  the  thermal  death-point  of  bacillus  typho- 
sus? 

Watch  the  demonstrator  use  the  anaerobic  culture 
tube. 

Widal's  Test  is  as  follows:  Take  one  drop  of  the 
blood  of  the  patient  and  mix  it  with  nine  drops  of 
water;  place  one  of  these  drops  on  a  cover-glass; 
make  ready  a  hanging-drop  slide;  now  transfer  one 
drop  of  a  culture  of  bacillus  typhosus  and  examine 
at  once.  If  an  agglutination  occurs  within  fifteen 
minutes  the  patient  has  typhoid  fever. 

Make  a  spread  of  bacillus  anthracis  and  examine 
it,  using  the  oil  lens.  Study  spore- formation  from  this 
bacillus. 


190  LABORATORY  METHODS  OF 

What  is  the  theory  of  immunity  ? 

Express  your  opinion  on  the  subject. 

Give  the  theory  of  phagocytosis. 

Demonstrate  the  anaerobic  culture  tube. 

Experiments  on  Animals. — These  are  for  the  pur- 
pose of  demonstrating  the  pathogenesis  of  the  germ. 
Take  the  animal  and  put  it  in  Voge's  Holder,  head 
downward.  Take  an  antiseptic  solution,  sterilize  the 
part  selected  for  the  injection,  and  at  the  same  time 
sterilize  your  syringe  in  hot  water.  Now  remove  the  lid 
of  your  Petri  dish  and  draw  the  required  amount  of 
growth  in  the  syringe.  (The  syringe  is  graduated  in 
c.c.  and  i-io  of  c.c.,  so  that  you  can  get  a  definite 
amount.)  Inject  the  material  in  the  animal  and  put 
the  animal  in  the  cage  until  next  period.  Examine  the 
animal  and  see  if  it  shows  signs  of  disease. 

Taking  the  Temperature  of  an  Animal. — Take  the 
animal  and  place  it  in  the  Vogue,  head  downward ;  put 
the  thermometer  in  the  rectum,,  and  note  degree. 

Make  a  spread  of  the  material  on  the  table  and  stain 
with  fuchsin. 

Bacillus  Pyocyaneus  is  a  short  rod.  It  is  three  mi- 
crons in  length  and  has  one  flagellum;  does  not  form 
spores;  frequently  forms  chains  of  four  or  six; 
can  exist  with  or  without  oxygen.  It  -stains  well  with 


BACTERIOLOGY.  191 

the  ordinary  stains  and  will  not  retain  Gram's  stain. 
It  is  the  cause  of  green  pus  in  a  wound.  It  produces 
the  green  color  to  the  pus  of  old  sores. 

Stain  a  spread  of  bacillus  Flouresence  Liq.,  and 
note  the  likeness  of  the  two. 

Stain  the  dejecta  at  your  desk  and  make  out  the 
morphology  of  the  species,  and  try  to  differentiate  the 
colon  bacilli  from  the  typhoid  bacilli,  if  such  are  pres- 
ent. Consult  the  atlas  and  find  the  germs  which  are 
most  like 'the  ones  on  the  slide. 

What  lesson  of  practical  value  can  you  get  from  this 
experiment  ? 

How  do  you  determine  the  power  of  any  germicide  ? 
Note  the  process  in  your  notebook. 

How  would  you  get  the  material  to  the  city  bacteri- 
ologists ? 

Name  five  diseases  which  you  must  report  to  the 
city  authorities. 

How  would  you  make  an  examination  of  milk  in 
case  you  were  called  on  to  do  so? 

What  bacteria  are  most  likely  to  be  in  the  milk? 


REAGENTS 
AND  STAINS 


From  OSBURN'S  MANUAL. 


13 


TECHNIQ  REAGENTS  AND  STAINS. 


VEGETABLE  SECTION. 

NO.    I.      METHOD  FOR  STAINING  FRESH  VEGETABLE  SEC- 
TIONS. 

(1)  Apply  section  to  slide  and  add  rosanilin  violet, 
one  to  five  minutes. 

(2)  Wash  in  water  to  remove  excess  of  stain. 

(3)  Dry  with  blotting  paper  and  add  glycerine  to 
dehydrate. 

(4)  Remove  excess  of  glycerine  and  add  glycerine 
again  to  thoroughly  dehydrate. 

(5)  Wipe  off  excess  of  glycerine  and  add  xylol 
twice. 

(6)  Apply  to  cover-glass  a  drop  or  two  of  xylol- 
balsam,  and,  having  wiped  off  the  excess  of  xylol  from 
the  slide,  drop  it  gently  (balsam  down)  upon  the  sec- 
tion.    Then  apply    general    pressure    with    dissecting 
needle  to  spread  out  the  balsam. 

(7)  Label  and  keep  in  a  horizontal  position  until 
the  balsam  is  hardened. 

(195) 


196  TECHNIC, 

Vegetable  Sections. — To  stain  paraffin  or  celloidin 
sections  of  plant  structures,  the  methods  are  practically 
the  same  as  those  given  below  for  animal  sections. 

ANIMAL    SECTIONS. 

NO.   2.      CARMINE  METHOD  FOR  FREE  SECTIONS. 

1 I )  Apply  section  to  slide  and  wash  with  thirty-five 
per  cent  alcohol. 

(2)  Add  lithium  carmine  sufficient  to  cover  section, 
one  to  five  minutes. 

(3)  If  necessary,  remove  excess  of  stain  with  acid 
alcohol,  five  to  ten  seconds. 

(4)  Dehydrate  with  increasing  strength  of  alcohol 
— thirty-five  per  cent,  seventy-five  per  cent,  and  ninety- 
five  per  cent,  and  absolute. 

(5)  Wipe  off  excess  of  alcohol,  and  when  section  is 
partly  dry  add  creosote  to  clear  up,  five  to  ten  minutes. 

(6)  Wipe  off  excess  of  creosote  and  mount  with 
balsam. 

(7)  Centre  cover-glass  and  apply  pressure  to  spread 
out  the  balsam. 

(8)  Label    and    lay    aside  in    horizontal    position, 
cover-glass  up,  until  the  balsam  hardens. 


REAGENTS  AND  STAINS.  197 

NO.    3.       CARMINE)    METHOD     WITH     AFFIXED     PARAFFIN 
SECTIONS. 

1 I )  Apply  to  the  center  of  the  slide  a  thin  layer  of 
collodion-clove-oil  mixture. 

(2)  Center  and  attach  the  section,  applying  the  heat 
of  a  spirit  or  Bunsen  flame. 

(3)  Immerse  in  xylol  two  minutes  and  in  turpen- 
tine ten    minutes  to    remove    paraffin.     Sections    im- 
mersed in  turpentine  alone  should  remain  twenty  min- 
utes. 

(4)  Wash  with  alcohol,  decreasing  strengths,  using 
thirty-five  per  cent  alcohol  last. 

(5)  Apply  lithium  carmine,  one  to  ten  minutes. 

(6)  Remove  excess  of  stain  with  acid  alcohol. 

(7)  Dehydrate  with  alcohol,  increasing  strengths. 

(8)  Dry  and  clear  up  with  creosote,   five  to  ten 
minutes. 

(9)  Wipe  of  excess  of  creosote  and  mount  in  bal- 
sam. 

(10)  Center  cover-glass. 

( 1 1 )  Label  and  lay  aside  in  horizontal  position  until 
balsam  hardens. 


198  TECHNIC, 

NO.   4.     CARMINE  METHOD  FOR  AFFIXED  CEUXDIDIN  SEC- 
TIONS. 

(1)  Center   section  and  affix  with  collodion  mix- 
ture. 

(2)  Stain  with  lithium  carmine,  one  to  five  minutes. 

(3)  Remove  excess  of  stain  with  acid  alcohol,  five 
or  ten  seconds. 

(4)  Apply  seventy  per  cent  alcohol. 

(5)  Apply  eighty  per  cent  alcohol. 

(6)  Apply  ninety-five  per  cent  alcohol  a  few  sec- 
onds. 

(7)  Clear  up  with  creosote. 

(8)  Remove  excess  of  creosote  with  blotting  paper. 

(9)  Mount  with  balsam  and  center  cover-glass. 

(10)  Label  and  lay  aside  in  a  horizontal  position. 

NO.    5.      HAEMATOXYUN    METHOD   -FOR   FREE   SECTIONS. 

1 I )  With  section  on  slide,  apply  alcohol  of  the  same 
strength  as  staining  solution. 

(2)  Stain  with  diluted    hsematoxylin,    one    to    ten 
minutes. 


REAGENTS  AND  STAINS.  199 

(3)  Remove   excess   of   stain  with  thirty-five  per 
cent  alcohol. 

(4)  Dehydrate  with  alcohols,  increasing  strengths. 

(5)  Clear  up  with  creosote  or  cedar  oil. 

(6)  Center  section  and  apply    balsam    and    cover- 
glass. 

(7)  Center  cover-glass. 

(8)  Label  and  lay  aside  in  horizontal  position  until 
balsam  hardens. 

NO.  6.     HAEMATOXYUN  METHOD  FOR  AFFIXED  SECTIONS. 

(1)  Apply  to  slide  a  thin  layer  of  egg-albumen  and 

glycerine. 

(2)  Center  section  and  flatten  it  by  gently  touching 
with  end  of  finger. 

(3)  Apply  heat  of  flame  until  paraffin  melts   (sec- 
tions that  have  been  flattened  upon  water  should  be 
heated  much  longer  than  others). 

(4)  Remove  paraffin  with  xylol  or  turpentine. 

(5)  Remove   xylol,    etc.,   with   alcohol,    decreasing 
strengths. 

(6)  Stain   with    diluted   hsematoxylin,    one   to   ten 
minutes. 


200  TECHNIC, 

(7)  Remove  excess  of  stain  with  thirty-five  per  cent 
alcohol. 

(8)  Dehydrate  with  alcohol. 

(9)  Clear  up  with  creosote,  five  to  ten  minutes. 

(10)  Wipe  off  excess  of  creosote  and  mount  with 
balsam. 

(u)   Center  cover-glass. 

(12)  Label   and   keep   in  horizontal  position  until 
balsam  hardens. 


NO.    7.       HAEMATOXYLIN    METHOD   FOR   AFFIXED 
DIN    SECTIONS. 


(  I  )  Center  section  and  affix  with  collodion  mixture. 

(2)  Stain  with  diluted  hsematoxylin,  one  to  ten  min- 
utes. 

(3)  Remove  excess  of  stain  with  acid  alcohol. 

(4)  Apply  seventy  per  cent  alcohol. 

(5)  Apply  eighty  per  cent  alcohol. 

(6)  Apply  ninety-five  per  cent  alcohol  a  few  sec- 
onds. 

(7)  Clear  up  with  creosote,  five  to  ten  minutes. 

(8)  Remove  excess  of  creosote  with  blotting  paper. 

(9)  Mount  with  balsam  and  center  cover-glass. 


REAGENTS  AND  STAINS.  201 

(10)  Label  and  lay  aside  in  a  horizontal  position 

until  balsam  is  hardened. 

/ 

NO.     8.       HA£MATOXYUN-£OSIN     METHOD. 

(1)  If  desired,  affix  section  to  slide  with  egg-albu- 
men and  glycerine  or  collodion  and  clove-oil. 

(2)  If  a  paraffin  -section,  remove  paraffin  with  xylol 
or  turpentine  or  both ;  remove  xylol,  etc.,  with  alcohol. 

(3)  Apply  thirty-five  per  cent  alcohol. 

(4)  Stain  with    diluted    hsematoxylin,    one  to  five 
minutes. 

(5)  Apply  thirty-five  per  cent  alcohol  to  remove  ex- 
cess of  stain. 

(6)  Stain  with  alcoholic  eosin  about  five  minutes. 

(7)  Apply  ninety-five  per  cent  alcohol  to  remove  ex- 
cess of  stain  and  dehydrate. 

(8)  Clear  up  with  cresote. 

(9)  Remove  excess  of  creosote  and  mount  with  bal- 
sam. 

(10)  Center  cover-glass  and  label. 

(n)   Lay  aside  in  horizontal  position  until  balsam 
hardens. 

Staining   Unicellular  Organisms. 
It  is  often  desirable  to  examine  materials  \\ithout 
staining.     This  is  accomplished  by  placing  upon  the 


202  TECHNIC, 

glass  slip  a  drop  of  the  material  tc  be  examined  and 
applying  cover-glass.  A  hair  placed  under  the  cover 
glass  will  prevent  the  object  from  being  crushed  and 
allow  of  free  motion  in  the  case  of  living  organisms. 
Should  it  be  desired  to  stain  such  preparations,  two 
methods  may  be  pursued,  irrigation  and  cover-glass 
staining. 

NO.    9.      IRRIGATION    AND   STAINING    MICRO-ORGANISMS. 

(1)  Place  upon  the  slide  a  drop  of  material  to  be 
studied. 

(2)  Apply  cover-glass. 

(3)  At  the  edge  of  the  cover-glass,  by  means  of  a 
pipette,  place  a  drop  or  two  of  the  reagents  or  stain. 

(4)  By  means  of  a  triangular  piece  of  blotting  paper 
applied  at  the  opposite  edge  of  the  cover-glass,  absorb 
the  moisture  from  the  preparation,  thus  drawing  under 
the  stain. 

NO.    10.      COV£R-GI,ASS  PREPARATIONS. 

(1)  Make  a  thin  spread  of  the  substance  to  be  ex- 
amined upon  a  sterilized  cover-glass. 

(2)  Using  a  Cornet  forceps,  dry  the  preparation  by 
holding  it  between  the  fingers  above  the  flame. 


REAGENTS  AND  STAINS.  203 

(3)  When   dry    pass  the    cover-glass    three    times 
through  a  flame,  keeping  the  preparation  up. 

(4)  Apply  stain. 

(5)  Wash  in  distilled  water  by  dipping  the  covier- 

glass  in  the  water  two  or  three  times. 

(6)  Examine  as  a  water  mount  or,  if  desired,  dry 
and  mount  in  balsam. 

(7)  Label  and  lay  aside  in  a  horizontal  position  until 
balsam  hardens. 

Note. — The  above  method  may  be  used  for  all  sim- 
ple staining.  Special  methods,  however,  are  often  used* 
and  they  will  be  given  as  required. 

LABORATORY  EXERCISE;. — Centering  and  labeling. 
Upon  the  under  side  of  your  box-cover  make  an 
outline  of  a  slide.  The  pencil  should  have  a  needle 
point.  Connect  opposite  angles  and  place  over  the  in- 
tersection of  the  lines  a  cover-glass.  Be  sure  that  the 
centre  of  the  cover-glass  coincides  with  the  center  of 
the  diagram.  Now,  carefully  make  an  outline  of  the 
cover-glass.  This  outline  may  be  used  for  centering 
both  the  sections  and  the  cover-glass.  Make  a  draw- 
ing of  this  outline  and  also  a  drawing  of  a 
slide  with  labels  and  cover-glass  in  situ.  Fill  in  the 
forms  of  labels  in  second  diagram,  using  the  following 
data :  A  transverse  section  of  the  muscle  of  a  normal 


204  TECHNIC, 

cat  was  stained  with  lithium  carmine  and  mounted  in 
balsam  in  October  i,  1891,  by  John  Smith. 

Drawings.  For  this  work  the  student  should  pro- 
vide himself  with  a  No.  5  or  a  No.  6  H  Faber  pencil, 
a  small  rule  or  triangle  and  a  sheet  of  thin  blotting 
paper.  The  pencil  should  be  kept  sharpened  to  a 
needle  point.  The  majority  of  students  will  say:  I 
cannot  draw.  An  honest  and  faithful  effort  will  often 
produce  gratifying  results.  Let  every  line  mean  some- 
thing. Be  scrupulously  neat  in  all  your  work.  Re- 
member that  this  work  will  furnish  a  better  exhibit  of 
character  and  ability  than  any  other  task  of  the  labora- 
tory. 

Abbreviations.  The  following  abbreviations  are  em- 
ployed in  this  text: 

Transverse  section — T.  S. 
Longitudinal  section — L.  S. 
Vertical  section— V.  S. 
Low  power — L.  P. 
High  power — H.  P. 
Cubic  centimeter — c.  c. 
Micro-millimeter — u. 
Millimeter — mm. 
Grami — g. 


REAGENTS  AND  STAINS.  205 

REAGENTS  AND  STAINS. 

In  preparing  the  following  regeants  it  is  well  to  re- 
member that  the  weight  of  a  cubic  centimeter  of  water 
is  one  gram,  and  that  a  liter  contains  1,000  cubic  centi- 
meters. The  formulae  that  are  given  are  those  most 
commonly  used  and  are  briefly  stated : 

NORMAL,  FLUIDS. 

Distilled  imter.- — A  supply  of  distilled  water  should 
be  constantly  at  hand  for  the  preparation  of  the  re- 
agents and  stains. 

Normal  saline. — This  is  prepared  by  dissolving  one 
part,  by  weight,  of  sodium  chloride  in  150  parts  of  dis- 
tilled water. 

MACERATING   FLUIDS. 

Dilute  alcohol. — This  may  be  prepared  by  mixing 
one  part  of  ninety-five  per  cent  alcohol  with  two  parts 
of  distilled  water.  Other  fluids  used  for  this  purpose 
are  solutions  of  potassium  bi-chromate,  two  per  cent, 
and  caustic  potash,  twenty-five  per  cent. 

DECALCIFYING  FLUIDS. 

Picric  acid. — Make  a  saturated  aqueous  solution  of 
picric  acid.  This  is  an  excellent  fluid  for  decalcifying 


206  TECHNIC, 

bones,  serving  at  the  same  time  as  a  staining  reagent. 
Crystals  should  be  added  from  time  to  time,  so  that 
some  undissolved  crystals  will  always  remain  in  the 
bottom  of  the  vessels. 

Nitric  acid. — Use  a  ten  per  cent  volumetric  solution 
in  water.     Decalcifkation  occurs  in  five  to  ten  days. 

FIXING  REAGENTS. 

Absolute  alcohol. — Specimens  should  remain  in  this 
reagent  from  one  to  six  hours,  according  to  size. 

Perenyi's  fluid — 

Nitric  acid  (ten  per  cent) 40  cc. 

Chromic  acid   (0.5  per  cent) 30  cc. 

Alcohol   30  cc. 

A  good  reagent  for  embryos  and  adult  tissues.  Time, 
three  to  twelve  hours ;  dehydrate  with  alcohol. 

Erlicki's  fluid- — 

Potassium  bi-chromate 2.5  grams. 

Cupric  sulphate  0.5  grams. 

Water   100  cc. 

Aqueous  Solution — 

Corrosive  Sublimate — 

Corrosive  sublimate I  gram. 

Water 95  cc. 


REAGENTS  AND  STAINS.  207 

Alcoholic  Solution — 

Corrosive  sublimate I  gram. 

Alcohol  (95  per  cent) 99  cc. 

Used  for  special  purposes  and  specially  foi  alimen- 
tary tract.  Time,  twenty-four  hours,  hardening  in  al- 
cohol, to  which  a  few  crystals  of  iodine  have  been 
added. 

Chromic  acid. — Use  a  0.5  per  cent  solution,  dehy- 
drating with  alcohol  in  the  dark. 

Muller's  fluid— 

Potassium  bi-chromate   ....  25  grams. 

Sodium  sulphate  10  grams. 

Water    1,000  cc. 

Pulverize  the  solids  before  adding  water,  and  use  a 
piece  of  camphor  in  the  solution  to  prevent  the  forma- 
tion of  fungi.  Good  for  general  use  and  especially  val- 
uable for  central  nervous  system.  Requires  from  two 
to  six  weeks.  Wash  in  water  for  several  days  and  de- 
hydrate with  alcohol. 

Plemming's  fluid — 

Chromic  acid  (one  per  cent  solution). 
46  cc. 


208  TECHNIC, 

Osmic  acid  (two  per  cent  solution) 

12   CC. 

Glacial  acetic  acid 3  cc. 

Especially  valuable  for  delicate  tissues.  Time, 
twenty-four  hours ;  dehydrate  with  alcohol. 

HARDENING   REAGENTS. 

Midler's  fluid,  corrosive  sublimate  solution,  chro- 
mic acid,  and  others  of  the  reagents  named  above  may 
be  used  for  hardening  purposes.  For  general  use,  iai/- 
cohol  will  be  found  invaluable.  The  tissue  should  be 
passed  through  increasing  strengths  of  alcohol,  seventy 
per  cent,  eighty  per  cent,  ninety  per  cent  ninety-five 
per  cent,  and  absolute.  It  should  be  allowed  to  re- 
main twenty- four  hours  in  each,  except  that  one  to  six 
hours  will  suffice  for  absolute  alcohol.  Ethyl  alcohol 
should  be  used,  or,  in  lieu  of  this,  methyl  alcohol 
makes  a  good  substitute.  To  prepare  absolute  alcohol, 
dehydrated  copper  sulphate  may  be  added  to  ethyl  or 
methyl  alcohol.  This  will  absorb  the  water  present. 

EMBEDDING   MEDIA. 

Paraffin  and  celloidin  are  extensively  used  for  em- 
bedding tissue. 


REAGENTS  AND  STAINS.  209 

FIXATIVES. 

Collodion  and  clove  oil  mixture. — Mix  one  part  of 
collodion  with  three  parts  clove-oil. 

Egg-ulbumen  and  glycerine. — Filter  the  whites  of 
several  eggs  and  add  to  the  filtrate  an  equal  volume  of 
glycerine.  To  the  mixture  add  a  few  drops  of  carbolic 
acid  or  a  small  piece  of  thymol  to  prevent  putrefaction. 

PARAFFIN    SOLVENTS. 

Xylol,  turpentine,  chloroform,  and  benzole  are  com- 
monly used  to  remove  paraffin  from  sections.  A  gooci 
plan  is  to  immerse  the  slide  containing  the  section  for  a 
few  moments  in  xylol,  and  then  transfer  to  turpen- 
tine for  ten  minutes. 

STAINING    SOLUTIONS. 

The  following  staining  preparations  are  those  most 
frequently  used,  and  will  be  found  adequate  to  the 
work  required  in  this  text.  Should  others  be  needed, 
the  formulae  can  be  obtained  from  more  advanced 
works. 

Hanstein's  rosanilin  violet — 

Methyl  violet I  gram. 

Fuchsin   I  gram. 

Distilled  water 100  cc. 

14 


210  TECHNIC, 

Note. — This  is  a  valuable  stain  for  vegetable  sec- 
tions. It  should  be  diluted  for  use  as  desired. 

Lithium  carmine — 

Carmine 2.5  grams. 

Lithium  carbonate  (saturated 

solution) 100  cc. 

The  carmine  should  be  dissolved  in  cold  solution. 
Sections  stain  rapidly,  and.  should  be  decolorized  with 
acid  alcohol. 

D ala-field's  haeinatoxylin — 

1.  Hsematoxylin I  gram. 

2.  Absolute   alcohol 6   cc. 

3.  Amonium  alum  (saturated 

sol.)    6  cc. 

4.  Glycerine   25  cc. 

Methyl  alcohol   .  .  ; 25  cc. 

Process. — Dissolve  (i)  in  (2);  add  this  solution  to 

(3)  ;  expose  to  air  and  light  for  a  week;  filter  and  add 

(4)  and  (5)  ;  allow  it  to  stand  for  a  long  time  exposed 
to  air  and  light. 

Alcoholic  Eosin  for  Sections. — Make  a  saturated  al- 
coholic solution.  This  is  used  as  a  ground  stain  in 
connection  with  hsematoxylin ;  also  as  a  blood  stain. 


REAGENTS  AND  STAINS.  211 

Magenta  for  blood,  etc. — 

Magenta i  gram 

•Alcohol  (eighty-five  per  cent). 50  cc. 

Water    150  cc. 

Glycerine   200  cc. 

Meythelene  blue  for  blood — 
Make  a  saturated  aqueous  solution. 
Carter's  carmine  mass  for  injecting — 

Carmine   3  grams 

Strong  ammonia 6  cc. 

Glacial  acetic  acid 6  cc. 

Coignet's   French   gelatin ...   7  grams 
Water   80  cc. 

Process. — "Place  the  finely  cut  gelatine  in  50  cc.  of 
water  for  five  hours ;  dissolve  the  carmine  in  mortar 
with  a  little  water  and  add  the  ammonia;  let  stand  for 
two  hours  and  then  pour  into  a  bottle,  rinsing  the  mor- 
tar with  the  remainder  of  water ;  place  the  gelatin  and 
water  on  a  water-bath  until  the  gelatin  melts.  To  the 
carmine  fluid  add  the  acetic  acid,  a  few  drops  at  a 
time  (rinsing  mortar  thoroughly)  until  the  fluid  be- 
comes crimson.  To  the  melted  gelatin  add  the  crim- 


212  REAGENTS  AND  STAINS. 

son  carmine,  little  by  little,  with  continual  stirring. 
Keep  in  a  cool  place  with  surface  covered  with  meth- 
ylated spirit.  When  wanted  for  use,  dissolve  on  water- 
bath  and  filter  through  flannel  wrung1  out  of  hot 
water."  (Fearnley's  Method.) 

CLEARING   AGENTS. 

Those  commonly  used  are  cedar  oil,  creosote,  clove- 
oil,  xylol,  and  aniline  oil.  Clove  oil  cannot  be  used 
with  celloidin  sections. 

MOUNTING   MEDIA. 

Glycerine  jelly  and  Canada  balsam,  are  commonly 
used  for  mounting  purposes.  For  the  laboratory,  bal- 
sam will  be  found  a  satisfactory  medium.  Should 
xylol  be  used  for  clearing,  the  balsam  should  be  dis- 
solved in  xylol.  Chloroform  balsam  may  be  used  in 
in  xylol.  Chloroform  balsam  may  be  used  in  sections 

For  the  formulae  of  reagents  and  stains  required  for 
work  in  bacteriology  and  urinalysis,  the  reader  is  re- 
ferred to  the  chapters  in  which  is  discussed  the  micro- 
technique of  these  subjects. 


MICROSCOPIC   TECHNIQUE. 

I.     REAGENTS  AND  STAINS. 

(l)    DECOLORIZING  SOLUTIONS. 

Twenty-five  per  cent  aqueous  solutions  of  hydro- 
chloric, nitric,  and  sulphuric  acids  may  be  used  for  de- 
colorizing. 

(2)  ACID  ALCOHOL. 

Hydrochloric  acid    I  part 

Alcohol  (seventy  per  cent) 100  parts 

(3)  IODINE  SOLUTION. 

Iodine I  gram 

Potassium  iodide 2  grams 

Water 90  cc. 

(4)  CARBOL   tfUCHSIN. 

Fuchsin I  cc. 

Alcohol 10  cc. 

Dissolve  and  add  100  c.c.  of  five  per  cent  solution  of 
carbolic  acid.  Filter. 

(213) 


214  TECHNIC, 

(5)   ACID  METHYLENE  BLUE. 

Sulphuric  acid 16  cc. 

Water    90  cc. 

Methylene  blue 2  grams 

This  stain  should  be  prepared  fresh  from  time  to 
time.     The  carbol  fuchsin  improves  with  age. 

(6)  LOFFLER'S  ALKALINE  METHYLENE  BLUE. 

Concentrated    alcoholic    solution    of 
methylene  blue  30  c. 

Potassium  hydrate   (aqueous  solu- 
tion  i-ioooo)    : 100  cc. 

This  is  especially  useful  in  staining  the  bacillus  of 
diptheria. 

(7)    ANILINE-WATER  GENTIAN  VIOLET. 

Aniline  oil   5  cc. 

Water 100  cc. 

Mix,  shake  vigorously,  filter;  the  fluid  after  filtra- 
tion should  be  perfectly  clear;  add 

Alcohol   10  cc. 

Alcoholic  solution  of  gentian  violet,  n  cc. 

This  solution  should  be  freshly  prepared  about  every 
two  weeks. 


REAGENTS  AND  STAINS.  215 

(8)  LQFFLER'S  MORDANT  FOR  FLAGELLA. 

Tannic   acid 2   grams. 

Water 8  cc. 

Saturated  solution  of  ferrous  sul- 
phate     5   cc. 

Saturated   alcoholic   solution   of 
fuchsin    I   cc. 

(9)    ANILINE-WATER  DYE  FOR   STAINING  SPORES. 

Saturated     alcoholic    solution    of 
fuchsin  or  gentian  violet ....   1 1  parts 

Aniline  oil  water 100  parts 

Abs.   alcohol    10  parts 

Keeps  well  for  ten  days. 

(lO)    AQUEOUS  STAINS. 

Saturated  aqueous  solutions  of  fuchsin,  gentian  vio- 
let, and  methyline  blue  will  be  found  useful  for  all 
simple  staining. 

(ll)   ALCOHOLIC  SOLUTIONS. 

Saturated  alcoholic  solutions  of  fuchsin,  gentian- 
violet,  and  methyline  blue  should  be  kept  on  hand  to 


216  TECHNIC, 

be   used  in   simple   staining   and   in   connection  with 
other   stains. 

II.     STAINING   METHODS. 

(1)  SIMPLE   STAINING. 

This  consists  in  using  a  single  stain. 

(2)  DOUBLE   STAINING. 

This  consists  in  using  two  stains,  one  to  stain  spores, 
protoplasm,  etc.,  and  the  other  as  a  ground  stain.  The 
following  methods  will  illustrate  double  staining: 

STAINING  OF  SPORES. 

(a)  Make  a  cover-glass  spread,  dry  and  pass  three 
times  through  the  flame. 

(b)  Add  aniline- water  gentian -violet. 

(c)  Heat  until  the  preparation  begins  to  boil;  re- 
move for  a  minute.     Repeat  this  process  six  times. 

(d)  Wash  in  three  per  cent  hydrochloric  acid-alcohol 
one  minute. 

(e)  Wash  in  water. 


REAGENTS  AND  STAINS.  217 

(f)   Counter- stain  with  aqueous  methylene  blue  half 
a  minute. 

(g),  Wash  in  water,       . 

(h)   Dry  and  clear  up  with  xylol. 

(i)   Mount  in  balsam. 


STAINING,  OF 

(a)  Mix  upon  the  cover-glass  a  portion  of  the  cul- 
ture with  a  drop  of  water,  using  care  not  to  break  off 
the  delicate  flagella. 

(b)  Dry  and  pass  three  times  through  a  flame. 

(c)  Apply  Loffler's  mordant  one  minute,  warming 
gently. 

(d)  Wash  in  water. 

(e)  Stain  with  aniline-water  fuchsin. 

(f )  Wash-  in  water.    , 

(g)  Dry  and  mount  in  balsam. 

GRAM'S  METHOD  FOR  BACTERIA. 

(a)  Make  a  cover-glass  preparation  by  the  usual 
method. 

(b)  Stain    with    aniline- water  gentian-violet    solu- 
tion, two  to  five  minutes,  warming  slightly. 

(c)  Add  Gram's  iodine  solution  one  and  one-half 
minutes. 


218  TECHNIC, 

(d)  Apply  alcohol,  repeatedly,  long  as  stain  contin- 
ues to  come  away  from  the  preparation. 

(e)  Wash  in  water  and  examine  as  a  water  mount. 

(f)  If  desired,  dry  and  mount  in  balsam. 

GABBETT'S  METHOD  FOR  TUBERCULOSIS,  ETC. 

(a)  Make  a  cover-glass  smear  of  the  sputum,  pus, 
blood,  or  urine  to  be  examined.     After  the  preparation 
is  dry,  affix  by  passing  three  times  through  the  flame. 

(b)  Using  a   Cornet   forceps,   apply  carbol-fuchsin 
five  to  ten  minutes,  heating  until  steam  appears. 

(c)  Wash  in  water. 

(d)  Apply  acid  methylene  blue  for  one  minute. 

(e)  Wash  in  water. 

(f)  Dry  and  mount  in  balsam. 

Staining  Tissues  for  Bacteria. 

Tissues  may  be  stained  in  Gram's  method  by  the  fol- 
lowing process : 

METHOD  FOR  STAINING  BACTERIA  IN  SECTIONS. 
IN    SECTIONS. 

(a)  Using  an  aqueous  solution  of  fuchsin,  gentian- 
violet  or  methylene  blue;  apply  stain  for  about  five 
minutes. 

(b)  Wash  in  water. 


REAGENTS  AND  STAINS.  219 

(c)  Apply  an  aqueous  solution  of  acetic  acid,  one 
per  cent,  for  one  minute. 

(d)  Apply  alcohol  two  minutes. 

(e)  Clear  up  with  xylol. 

(f)  Mount  with  balsam. 


INDEX. 


Abbe's  condenser,  15,   17 
Accessories     to     the     micro- 
scope,  15 
Acetic    acid    action    on   cells, 

21,   53,   54 
Acids  stains,  see  technic  and 

stains 

Adenoid  tissue,  66 
Adipose  tissue,  41 
Alcohol  as  a  hardening  agent, 

29 

Alveolar  ducts,  121 
Alveolus,    121 
Amoeba,  26 

Amoeboid   movement,   53 
Analyzer,  17 
Animalcule,    23 
Anterior  chamber  of  the  eye, 

124 
Anterior     column     of     cord, 

77 

Anterior  cornua,  75 
Apical  foramen,  94 
Aqueous  humor,  125 
Arachnoid,  77 
Areas  of  the  cord,  76 
Arrectores     muscle     of     the 

hair,  89 
Arteries,  61 

coats  of,  63 
Axis    cylinder,    73 
Axilemma,    73 
Animal  autopsies  on,  174 

inoculation,  170,  175 
Anthrax,  160 
Antiseptics,  170 


Attenuation  of  germs, 

Bacilli,  182 
Bacteria,  159,  177 

agglutination  test,  183,  189 

anaerobes,  190 

biology  of,  184 

classification  of  159 

cultivation  of,  165 

death  of  169 

food  supply  of,  159 

microscopic  examination  of, 
173 

movement  of,  164 

parasitic,  165 

saprophytic  bacteria,  165 

spores  of,  176 

temperature  of  growth,  160, 

162 

Bacteriological  diagnosis.  186 
Bacteriology,  159 
Bartholin's  glands,  119 
Basement    membrane,    96 
Basophiles,   53 
Bellin's  tube,  110 
Bile  capillaries,  104 

duct,  104 
Bladder,   113 
Blood  tissue,  49 

platelets,    49 

corpuscles,  49 

plasma,  49 

shadows,  53 

origin    of,    53,    55 

crystals,    53 
Bone,  45 

(221) 


222 


INDEX. 


canaliculi,   46 

cells,    46 

development  of,  47 

blood   vessels,    47 
Bronchi,   120 
Bronchiole,   120 
Brownian  movement,  32 
Brunner's  glands,  99 
Budding,  18 
Bulb  hairs,  90 
Bacteria,  159 
Bouillon,  179 

Camera  lucida,  15 
Capillaries,   61 

bile,    104 

lymph,   65 
Capsule  of  Glisson,  102 

of  glands,  105 

of  lymph  glands,  66 
Cardiac  muscle-cells,  58 
Carmine    stain,    see    chaptei 

on   stains   and   reagents 
Cartilage,  43 

capsule  of,  44 

fibro-elastic,    45 

hyaline,   44 

lacunae  of,  44 

matrix  of,   44 
Cells,  18,   19 

blood  cells,  49 

bone,    46 

cartilage,   44 

of  stomach,  96 

ciliated,   36 

connective-tissue 

containing  pigment,  86 

of  cerebellum,  82 

double    staining   of    blood, 
55 

eosinophile,  53 

epithelial,    35 

fat,  41 

fixing   of,    30 

ganglion,  73 

germinal,   117 


gland,  70 

goblet,  98,  101 

muscle,   56 

nerve,   72 

neuroglia,  72 

of  Purkinje,   83 

olfactory,   123 

polynuclear,  53 

pyramidal   large,   82 

small,   82 

spindle,  81 
Cell  division  direct,  29 

indirect,   29 
Cellodin,   see  the  chapter  on 

technic 

Cementum,   93 
Centrosomes,    28 
Chondrin,  44 
Choroid,  124 
Ciliated  cells,  36 
Circulatory  system,  61 
Circumvallate  papillae,   127 
Clitoris,   119 

Coil-glands  of  the  skin,  87 
Commissures,  76,  78 
Compound   microscope,    11 
Condenser,  15 
Connective  tissue,  38 
Convoluted  tubule,  114 
Corium,   89 
Cornea,  124 
Corpus  luteum,  118 
Cortical  layer  of  hair,  88 

nodule  of  lymphatic  gland, 
100 

substance  of  kidney,   10 
Cover-glass,  16 
Cowper's  gland,  114 
Crystalline  lens,  125 
Cutical,    85 
Cutis,  85 

Carbol-fuchsin,  188 
Cholera,  186 
Cocci,  159 

Colony  counting,  168 
Culture  media,  160 


INDEX. 


223 


hanging-drop,  167,  175 
incubation  of, 
plate,  174 
shake,  174 

Decalcifying  fluid,  see  technic 
Delafield's    haematoxylin, 
Dendrites,   72 
Dentine,  93 
Dentinal   bulbs,   95 
Diaster,    29 

Direct  cell-division,  29 
Discus  proligerous,  118 
Dura  mater,  77 

Ear,  125 
Ectoderm,    34 
Elastic  fibers,  45 
Embedding  media, 
Enamel,  93 
Endocardium,  61 
Endomysium,  58 
Emd-organ,  56 
Epimysium,    58 
Epithelium,   35 

cut  of,  35 
Esophagus,   95 
Exoplasm,   36 
Eye,  124 

Fallopian  tubes,  118 

Fat  cells,  41 

Female  generative  organs,  16, 

17,  18,  19 

Fenestrated  membrane  63 
Fibro-cartilage,  45 
Filiform   papillae,   127 
Flagella,  see  cilia 
Fungiform  papillae,  127 
Flagella,  176,  182 

Ganglia,  73 

Genital  organs  of  male,  114 
Germ  center  of  lymphoid  tis- 
sue, 68 
Germinal  cells  of  ovary,  118 


spot,   118 

vesicle,  118 
Gonococcus,  188 
Gram's  method,  172,  176 
Glands,  type  of,  70 

structure  of,  71 

submaxillary,  106 
Glisson's  capsule,   102 
Glomerulus,  110 
Goll's  column,  77 
Gower's    haemacytometer,    50 
Graafian  follicle,  118 
Gray  matter,  77 

Hair,  87 

shedding  of,  90 
Hassal's  corpuscles,  69 
Haversian  canal,  46 
Heart,   61 
Haematin,   54 
Haematoidin,    54 
Haemin,  54 
Haemoglobin,  54 
Hilum  of  lymph  glands,  68 
Histology,  general,  20,  26 
Hyaline  cartilage,  44 

Immersion  lens, 

Incubate,  168 

Immunity,  190 

Indifferent  fluid, 
salt  solution,  22 

Indirect  cell-division,  29 

Infiltration, 

Infiltrated,  30 

Influenza,  188 

Injection    fluids 
gelatin-carmin 

Inoculation  of  animals,  174 

Intermoleciilar    layer    of    ret- 
ina, 125 

Interglobular  spaces,  93 

Interglobular  artery,  103 
vein,  103 

Intestines,  97 
large,  101 


224 


INDEX. 


mucous    membrane    of,    see 

the  stomach 
nerve  supply,  100 
small,  97 
technic,  100 
Intralobular  artery  of  kidney 

112 

vein  of,  112 
Iris  diaphragm,  15 

Kidney,  109 

arched     collecting     portion 

of  tubule,  112 
blood  vessels  of,  110 
cortex  of,  109 
distal  convoluted  tubule  of, 

112 

medullary  substance  of,  110 
straight    collecting    tubule, 

112 

Knife  of  microtome,  77 
Krause's  membrane,  58 

Lacunae  of  bone,  46 
Lamellae  of,  46 
Large  intestines,  101 
Lateral  columns  of  the  cord, 

77 

Layer  of  Henle,  88 
Leucocytes,  see    lymphocytes 
Lens,  15 

Abbe,  15 

of  the  eye,  125 
Lieberkuhn's    glands,   99 
Ligaments  of  the  eye,  125 
Lingual     mucous     membrane, 
126 

papillae,  127 
Liquor  folliculi, 
Liver,  102 

vascular  system  of,  103 
Lobules  of  liver,  102 
Loop  of  Henle,   112 
Lungs,   120 

lymphatics  of,  121 
Lymph  glands,  67 


capillaries,  65 

follicles,  66 

of  the  tongue,  126 

solitary  glands,  100 
Lymphocytes,  53 
Lymphoid  tissue 
Lymph-vessels,   65 

Male  generative  organs,  114 
Malpighian   corpuscles,   68 
Mammary  gland,  90 
Matrix;    44 

of  bone,  46 

of  nail,  87 

Mediastinum  testis,  115 
Medullary  sheath,  77 
Medullated  nerve  fibers,  74 
Membrana  propria, 
Mesoderm,  34 

Microscope    and   its   accesso- 
ries, 11 

coarse  adjustment  of,  12 

compound,  11 

description  of,  11 

fine  adjustment,  12 

parts  of,  11 

simple,  11 

study  of,  12 
Microtome,  16 

student's   18 
Milk,  91 

Mitosis,     see    indirect   cell-di- 
vision 

Mixed  glands,  105 
Molecular  movement  of  cells, 

see  Brownian   movement 
Mononuclear  cells,  53 
Morula,  33 
i  Mount, 

|  Mouth  glands  of,  107 
!  Mucous  glands,  105 
Muscle  cell,  56 

cardiac,  58 

heart,  58 

smooth,  see  involuntary,  58 

striped,  see  voluntary,  56 


INDEX. 


225 


fiber,  56 
tissue,  56 

Muscularis  mucosae  of  the  in- 
testines, 98 

Myocardium,  61 

Nails,  87 

bed,  87 

body,  87 

matrix,  87 
Nerves,  72 

end-organs,    56 

cells  of,  72 

fibers,  73 

medullated,  74 

non-medullated,  74 
Neurilemma,  73 
Neuroglia  cells,  72 
Neurone,  72 
Nitric  acid, 
Nodes  of  Ranvler,  73 
Nodules, 

lymph,  66 
Nuclear  membrane,  28 

stain 

Neucleolus,  28 
Nucleus,  28 

Objectives,  12 
Odontoblasts,  95 
Olfactory  tract,  123 
Ossification,  47 
Ovary,  117 

germinal     epithelial    layer, 
117 

medullar   and   cortical   sub- 
stance, 117 

blood  vessels  of,  118 
Ovum,  118 

Pacchionian  bodies,  77 
Pancreas,  108 
Papillae,  127 

circumvallate,  127 

filiform,  127 

fungiform,  127 
Paraffin, 
15 


Parotid  gland,  106 

Penis,   116 

Pericardium,  61 

Perichondrium,  44 

Perimysium,  58 

Periosteum,  47 

Pia  mater,  77 

Picro  carmin  as  a  stain,  see 
technic 

Pigment  layer  of  the  eye,  12b 

Plural  staining,  see  double  in 
the  technic 

Portal  vein,  103 

Primitive  jaw,  94 

Prostatic  portion  of  the  ure- 
thra, 113 

Protococcus,  20 

Protoplasm,  26 

Pseudopod,  26 

Pulp  of  spleen,  68 

Purkinje's  cell,  82 

Pyramidal  cells,  80 

Rectum,  102 

Red  blood  corpuscle,  51 
estimation  of,  50 

Respiratory  system,   120 

Retina,  124 

Respiratory  bronchiole, 
layers  of,  125 

Retzius,  93 

Rouleaux  of  red  blood  corpus- 
cles 

Salivary  glands,  105 

blood  supply,  105 

lymphatics  of,  105 

nerve  supply,   105 

scheme  of,  70 
Saprophyte,  182 
Sarcolemma,  56 
Sarcous  elements,  56 
Sclera,  124 
Sebaceous  glands,  87 
Section  staining,   see  technic 
Sectioning,  see  technic 


226 


INDEX. 


Sense  organs,  123 
Sensory  nerve  endings,  123 
Sharpey's  fibers,  47 
Sinus  of  kidneys,  113 
Skin  and  its  appendages,  85 

glands  of  the  skin,  87 

nerve  endings  of,  86 

pigment  of,  113 

structure  of,  85 
Slides,  16 

Slipper  animalcule,  23 
Solitary  lymph-follicle,  99 
Specimens  drawings  of,  5 
Spermatoblasts,    114 
Spermatozoa,  114 
Spermatozoon,  116 
Spinal  cord,  75 

anterior  median  fissure,  78 

gray  matter,  77 

horns  of,  78 

posterior  median  fissure,  78 

white  substance  of,  75 

ganglion  cells  of,  73 
Spindle   cells,    81 
Spirogyra,   22 
Spleen,  67 
Splenic  pulp,  68 
Spongioplasm,  26 
Stains,  see  technic 
Sterilization,  114,  161 
Sterilizers,  165' 
Stomach,  96 
Stomata,  69 

Stratified  epithelium,  126 
Streptococcus,  187 
Subarachnoid    space,    77 
Subdural  space,  77, 
Submaxillary  gland,  105 
Sudoriparous  glands,  87 
Suspensory  ligament,  125 
Sweat-glands,  87 
Sympathetic   gangla 

Taste-buds,  126 

Tease,  39 

Technic,  reagents  and    stains 


Teeth,  92 
structure  of,  92 

Tendon,  39 

Test-tubes,  162 

Theca  folliculi,  118 

Thymus  gland,  68 

Tongue,  126 

Tooth  pulp,  95 

Tubule 

uriniferous,  109 
straight  collecting,  112 
schematic  drawing  of,  111 
tunica  albuginea,  114 

Theory  of  immunity, 
phagocytosis,  190 

Tubercle  bacillus,  186 

Ureters,  113 
Urethra,  113 
Urinalysis,  129 

albumin,    14 

albuminometer    Esbach's, 
146 

alkaline    phosphate,    142 

apparatus     required     for 
urine   examination,    134 

bile,  153 

Gmelin's  test  for,  154 

blood  in,  154 

cherniqal  reaction  of,  140 

chlorides  in,  141 

color,  140 

composition   of,   140 

creatin,  141 

creatmin,  141 

Fehling's  solution,  152 

fermentation  test,  147 

haematuria,  154 

Heidenhain's  theory  as  to 
the  secretion  of  the  urine, 
133 

hydrobilirubin,    152 

introduction.  131 

kidney,   133 

Ludwig's  theory,   133 

mucin,    151 


INDEX. 


227 


nitric  acid  test,  145 
nucleoalbumin,  151 
odor  of  urine, 
peptones,  152 
phosphates,  142 
pus  in  the  urine,  154 
quantity  of,  140 
reaction  of  urine,   134 
reagents     required     in    the 

analysis  of  the  urine,  135 
secretion  of  urine,   133 
sediment  in  urine,  150 
selecting     a     specimen     of 

urine,  135 
specific  gravity  of  the  urine,  | 

140 

sugar  in  the  urine,  146 
sulphates  in  the  urine,  146, 
urates,  135 
urea,   135 
uroerythrin,  153 
Uterus,  118 
glands  of,  119 


Vacuole,  19 
Vagina,  119 

glands  of,  119 
Vasa  afferentia 

efferentia 

vas  deferens,  115 
Veins, 

intralobular,  102 

portal,  103 
Valve,  65 

rectal,   102 

Villi  of  small  intestines,  97 
Volkmann's  canal,  47 

Widal's  serum  diagnosis,  183 

Wandering  cells,  40 

White  blood  corpuscles,  53 

Xylol,   see  technic 
Yeast,  18 

Zona  pellucida,   118 
tissue  of,  120 


1903 


.QPYOELTOCAT.DW 
MAY    18 


-I  'y3 


UNIVERSITY  OF  CALIFORNIA  LIBRARY 


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